Regulatory nucleic acid molecules for enhancing constitutive gene expression in plants

ABSTRACT

The present invention is in the field of plant molecular biology and provides methods for production of high expressing constitutive promoters and the production of plants with enhanced constitutive expression of nucleic acids wherein nucleic acid expression enhancing nucleic acids (NEENAs) are functionally linked to the promoters and/or introduced into plants.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/393,028 filed Feb. 28, 2012, which is a national stageapplication (under 35 U.S.C. § 371) of PCT/EP2010/061659, filed Aug. 11,2010 which claims benefit of U.S. Provisional Application No.61/238,230, filed Aug. 31, 2009 and European Application No. 09169019.8,filed Aug. 31, 2009. The entire contents of each of these applicationsare hereby incorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_074021_0171_01. The size of thetext file is 95 KB and the text file was created on Oct. 30, 2017.

BACKGROUND OF THE INVENTION

The present invention is in the field of plant molecular biology andprovides methods for production of high expressing constitutivepromoters and the production of plants with enhanced constitutiveexpression of nucleic acids wherein nucleic acid expression enhancingnucleic acids (NEENAs) are functionally linked to said promoters and/orintroduced into plants.

Expression of transgenes in plants is strongly affected by variousexternal and internal factors resulting in a variable and unpredictablelevel of transgene expression. Often a high number of transformants haveto be produced and analyzed in order to identify lines with desirableexpression strength. As transformation and screening of lines withdesirable expression strength is costly and labor intensive there is aneed for high expression of one or more transgenes in a plant. Thisproblem is especially pronounced, when several genes have to becoordinately expressed in a transgenic plant in order to achieve aspecific effect as a plant has to be identified in which each and everygene is strongly expressed.

For example, expression of a transgene can vary significantly, dependingon construct design and positional effects of the T-DNA insertion locusin individual transformation events. Strong promoters can partiallyovercome these challenges. However, availability of suitable promotersshowing strong expression with the desired specificity is often limited.In order to ensure availability of sufficient promoters with desiredexpression specificity, the identification and characterization ofadditional promoters can help to close this gap. However, naturalavailability of promoters of the respective specificity and strength andthe time consuming characterization of promoter candidates impedes theidentification of suitable new promoters.

In order to overcome these challenges, diverse genetic elements and/ormotifs have been shown to positively affect gene expression. Amongthese, some introns have been recognized as genetic elements with astrong potential for improving gene expression. Although the mechanismis largely unknown, it has been shown that some introns positivelyaffect the steady state amount of mature mRNA, possibly by enhancedtranscriptional activity, improved mRNA maturation, enhanced nuclearmRNA export and/or improved translation initiation (e.g. Huang andGorman, 1990; Le Hir et al., 2003; Nott et al., 2004). Since onlyselected introns were shown to increase expression, splicing as such islikely not accountable for the observed effects.

The increase of gene expression observed upon functionally linkingintrons to promoters is called intron mediated enhancement (IME) of geneexpression and has been shown in various monocotyledonous (e.g. Calliset al., 1987; Vasil et al., 1989; Bruce et al., 1990; Lu et al., 2008)and dicotyledonous plants (e.g. Chung et al., 2006; Kim et al., 2006;Rose et al., 2008). In this respect, the position of intron in relationto the translational start site (ATG) was shown to be crucial for intronmediated enhancement of gene expression (Rose et al., 2004).

Next to their potential for enhancing gene expression, a few intronswere shown to also affect the tissue specificity in their nativenucleotide environment in plants. Reporter gene expression was found tobe dependent on the presence of genomic regions containing up to twointrons (Sieburth et al., 1997; Wang et al., 2004). 5′ UTR introns havealso been reported to be of importance for proper functionality ofpromoter elements, likely due to tissue specific gene control elementsresiding in the introns (Fu et al.,1995a; Fu et al., 1995b; Vitale etal., 2003; Kim et al., 2006). However, these studies also show thatcombination of introns with heterologous promoters can have strongnegative impacts on strength and/or specificity of gene expression(Vitale et al., 2003; Kim et al., 2006, WO2006/003186, WO2007/098042).For example the strong constitutive Cauliflower Mosaic Virus CaMV35Spromoter is negatively affected through combination with the sesameSeFAD2 5′UTR intron (Kim et al., 2006). In contrast to theseobservations, some documents show enhanced expression of a nucleic acidby IME without affecting the tissue specificity of the respectivepromoter (Schünmann et al., 2004).

In the present application further nucleic acid molecules are describedthat enhance the expression of said promoters without affecting theirspecificity upon functionally linkage to constitutive promoters. Thesenucleic acid molecules are in the present application described as“nucleic acid expression enhancing nucleic acids” (NEENA). Introns havethe intrinsic feature to be spliced out of the respective pre-mRNA. Incontrast to that the nucleic acids presented in the application at hand,do not necessarily have to be included in the mRNA or, if present in themRNA, have not necessarily to be spliced out of the mRNA in order toenhance the expression derived from the promoter the NEENAs arefunctionally linked to.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Luciferase reporter gene expression analysis in transientlytransformed A. thaliana leaf protoplasts of NEENA-less (LJK132) andNEENA-containing constructs (LJK91-LJK133) representing putative NEENAmolecules deriving from constitutively expressed genes under the controlof the p-AtNit1 promoter. Normalization was performed bycotransformation and analysis of the Renilla luciferase and expressionvalues are shown in relation to the NEENA-less control construct(LJK132=1). Expression values are shown in relation to the NEENA-lesscontrol construct (LJK134=1).

FIG. 2: Bar graph of the luciferase reporter gene activity shown asrelative light units (RLU) of independent transgenic oilseed rape plantlines harboring NEENA-less (LJK138) or NEENA-containing reporter geneconstructs representing NEENA molecules from constitutively expressedgenes (LJK139-LJK144) under the control of the p-AtNit1 promoter andafter normalization against the protein content of each sample (averagedvalues, tissues of 20 independent transgenic plants analyzed). A) leaftissue, B) flowers, C) siliques

FIG. 3: Bar graph of the luciferase reporter gene activity shown asrelative light units (RLU) of independent transgenic soybean plant linesharboring NEENA-less (LJK138) or NEENA-containing reporter geneconstructs representing NEENA molecules from constitutively expressedgenes (LJK139-LJK144) under the control of the p-AtNit1 promoter andafter normalization against the protein content of each sample (averagedvalues, tissues of 10 independent transgenic plants analyzed). A) leaftissue, B) flowers, C) seeds

FIG. 4: Bar graph of the luciferase reporter gene activity shown asrelative light units (RLU) (log scale) of independent transgenic maizeplant lines harboring NEENA-less (LJK309) or NEENA-containing reportergene constructs representing NEENA molecules from constitutivelyexpressed genes (LJK326-LJK327) under the control of the p-ZmUbipromoter and after normalization against the protein content of eachsample (averaged values, tissues of 15 independent transgenic plantsanalyzed). A) leaf tissue, B) kernels

FIG. 5: Bar graph of the luciferase reporter gene activity shown asrelative light units (RLU) of independent transgenic rice plant linesharboring NEENA-less (CD30963) or the NEENA-containing reporter geneconstruct representing a NEENA molecule from constitutively expressedgenes (CD30964) under the control of the constitutive p-PRO239 promoterand after normalization against the protein content of each sample(averaged values, tissues of 15 independent transgenic plants analyzed).A) leaf tissue, B) seeds

DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the invention comprises a method for production ofa high expression constitutive promoter comprising functionally linkingto a promoter one or more nucleic acid expression enhancing nucleic acid(NEENA) molecule comprising

i) the nucleic acid molecule having a sequence as defined in any of SEQID NO: 1 to 19, preferably SEQ ID NO: 1 to 9, or

ii) a nucleic acid molecule having a sequence with an identity of 80% ormore to any of the sequences as defined by SEQ ID NO:1 to 19, preferablySEQ ID NO: 1 to 9, preferably, the identity is 85% or more, morepreferably the identity is 90% or more, even more preferably, theidentity is 95% or more, 96% or more, 97% or more, 98% or more or 99% ormore, in the most preferred embodiment, the identity is 100% to any ofthe sequences as defined by SEQ ID NO:1 to 19, preferably SEQ ID NO: 1to 9 or

iii) a fragment of 100 or more consecutive bases, preferably 150 or moreconsecutive bases, more preferably 200 consecutive bases or more evenmore preferably 250 or more consecutive bases of a nucleic acid moleculeof i) or ii) which has an expressing enhancing activity, for example 65%or more, preferably 70% or more, more preferably 75% or more, even morepreferably 80% or more, 85% or more or 90% or more, in a most preferredembodiment it has 95% or more of the expression enhancing activity asthe corresponding nucleic acid molecule having the sequence of any ofthe sequences as defined by SEQ ID NO:1 to 19, preferably SEQ ID NO: 1to 9, or

iv) a nucleic acid molecule which is the complement or reversecomplement of any of the previously mentioned nucleic acid moleculesunder i) to iii), or

v) a nucleic acid molecule which is obtainable by PCR usingoligonucleotide primers described by SEQ ID NO: 20 to 57, preferably SEQID NO: 20/21; 26/27; 30/31; 38/39; 42/43; 44/45; 46/47; 50;51 and 56/57as shown in Table. 2 or

vi) a nucleic acid molecule of 100 nucleotides or more, 150 nucleotidesor more, 200 nucleotides or more or 250 nucleotides or more, hybridizingunder conditions equivalent to hybridization in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO4.1 mM EDTA at 50° C. with washing in 2×SSC,0.1% SDS at 50° C. or 65° C., preferably 65° C. to a nucleic acidmolecule comprising at least 50, preferably at least 100, morepreferably at least 150, even more preferably at least 200, mostpreferably at least 250 consecutive nucleotides of a transcriptionenhancing nucleotide sequence described by SEQ ID NO:1 to 19, preferablySEQ ID NO: 1 to 9 or the complement thereof. Preferably, said nucleicacid molecule is hybridizing under conditions equivalent tohybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTAat 50° C. with washing in 1×SSC, 0.1% SDS at 50° C. or 65° C.,preferably 65° C. to a nucleic acid molecule comprising at least 50,preferably at least 100, more preferably at least 150, even morepreferably at least 200, most preferably at least 250 consecutivenucleotides of a transcription enhancing nucleotide sequence describedby SEQ ID NO:1 to 19, preferably SEQ ID NO: 1 to 9 or the complementthereof, more preferably, said nucleic acid molecule is hybridizingunder conditions equivalent to hybridization in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 50° C. or 65° C., preferably 65° C. to a nucleic acidmolecule comprising at least 50, preferably at least 100, morepreferably at least 150, even more preferably at least 200, mostpreferably at least 250 consecutive nucleotides of a transcriptionenhancing nucleotide sequence described by any of the sequences asdefined by SEQ ID NO:1 to 19, preferably SEQ ID NO: 1 to 9 or thecomplement thereof.

In one embodiment, the one or more NEENA is heterologous to the promoterto which it is functionally linked.

As described above under v) the nucleic acid molecule obtainable by PCRusing oligonucleotides as defined by SEQ IDs 20 to 57, preferably SEQ IDNO: 20/21; 26/27; 30/31; 38/39; 42/43; 44/45; 46/47; 50/51 and 56/57 asshown in Table 2 is obtainable for example from genomic DNA fromArabidopsis plants such as A. thaliana using the conditions as describedin Example 1 below.

The skilled person is aware of variations in the temperature profile,cycle number and/or buffer composition or concentration to obtain therespective NEENA molecule. The specific combination of oligonucleotidesto be used in the respective PCR reaction for obtaining a respectiveNEENA molecule is described in Table 2.

A person skilled in the art is aware of methods for rendering aunidirectional to a bidirectional promoter and of methods to use thecomplement or reverse complement of a promoter sequence for creating apromoter having the same promoter specificity as the original sequence.Such methods are for example described for constitutive as well asinducible promoters by Xie et al. (2001) “Bidirectionalization of polarpromoters in plants” nature biotechnology 19 pages 677-679. The authorsdescribe that it is sufficient to add a minimal promoter to the 5′ primeend of any given promoter to receive a promoter controlling expressionin both directions with same promoter specificity. Hence a highexpression promoter functionally linked to a NEENA as described above isfunctional in complement or reverse complement and therefore the NEENAis functional in complement or reverse complement too.

A constitutive promoter as used herein means a promoter expressed insubstantially all plant tissues throughout substantially the entire lifespan of a plant or part thereof. A promoter expressed in substantiallyall plant tissues may also encompass promoters that are expressed in atleast two of the main plant tissues such as leaf, stem and/or root andmay or may not be expressed in some or all minor tissues or cells suchas epidermis, stomata, trichome, flower, seed or meristematic tissue. Ina preferred embodiment a constitutive promoter as meant herein isexpressed at least in green tissues such as leaf and stem.

A promoter expressed throughout substantially the entire life span of aplant or part thereof may also encompass promoters that are expressed inyoung and developed tissue but may lack expression at specific timepoints in the lifespan of a plant or under specific conditions such asduring germination and/or senescence or under biotic and/or abioticstress conditions such as fungi or bacterial infection, drought, heat orcold. In a preferred embodiment a constitutive promoter expressed insubstantially the entire lifespan of a plant is expressed at least infully expanded tissue until onset of senescence.

In principal the NEENA may be functionally linked to any promoter suchas tissue specific, inducible, developmental specific or constitutivepromoters. The respective NEENA will lead to an enhanced expression ofthe heterologous nucleic acid under the control of the respectivepromoter to which the at least one NEENA is functionally linked to. Theenhancement of expression of promoters other than constitutivepromoters, for example tissue specific promoters, will render thespecificity of these promoters. Expression of the nucleic acid undercontrol of the respective promoter will be detectable in additionaltissues or developmental stages the transcript of said nucleic acid hadnot been detected without the NEENA. Hence, tissue- or developmentalspecific or any other promoter may be rendered to a constitutivepromoter by functionally linking at least one of the NEENA molecules asdescribed above to said promoter. It is therefore another embodiment ofthe invention to provide a method for rendering the specificity of anygiven promoter functional in plant to a constitutive promoter by linkingthe respective promoter to a NEENA molecule comprising a sequence asdescribed above under i) to vi).

Preferably, the one or more NEENA is functionally linked to anyconstitutive promoter and will enhance expression of the nucleic acidmolecule under control of said promoter. Constitutive promoters to beused in any method of the invention may be derived from plants, forexample monocotyledonous or dicotyledonous plants, from bacteria and/orviruses or may be synthetic promoters. Constitutive promoters to be usedare for example the PcUbi-Promoter from P. crispum (WO 2003102198), theZmUbi-Promoter from Zea maize, AtNit-promoter from the A. thaliana geneAt3g44310 encoding nitrilase 1, the 34S-promoter from figwort mosaivvirus, the 35S-promoter from tobacco mosaic virus, the nos andocs-promoter derived from Agrobacteria, the ScBV-promoter (U.S. Pat. No.5,994,123), the SUPER-promoter (Lee et al. 2007, Plant. Phys.), theAtFNR-promoter from the A. thaliana gene At5g66190 encoding theferredoxin NADH reductase, the ptxA promoter from Pisum sativum(WO2005085450), the AtTPT-promoter from the A. thaliana gene At5g46110encoding the triose phosphate translocator, the bidirectionalAtOASTL-promoter from the A. thaliana genes At4g14880 and At4g14890, thePRO0194 promoter from the A. thaliana gene At1g13440 encoding theglyceraldehyde-3-phosphate dehydrogenase, the PRO0162 promoter from theA. thaliana gene At3g52930 encoding the fructose-bis-phosphate aldolase,the AHAS-promoter (WO2008124495) or the CaffeoylCoA-MT promoter and theOsCP12 from rice (WO2006084868).

The high expression constitutive promoters of the invention functionallylinked to a NEENA may be employed in any plant comprising for examplemoss, fern, gymnosperm or angiosperm, for example monocotyledonous ordicotyledonous plant. In a preferred embodiment said promoter of theinvention functionally linked to a NEENA may be employed inmonocotyledonous or dicotyledonous plants, preferably crop plant such ascorn, soy, canola, cotton, potato, sugar beet, rice, wheat, sorghum,barley, musa, sugarcane, miscanthus and the like. In a preferredembodiment of the invention, said promoter which is functionally linkedto a NEENA may be employed in monocotyledonous crop plants such as corn,rice, wheat, sorghum, musa, miscanthus, sugarcane or barley. In anespecially preferred embodiment the promoter functionally linked to aNEENA may be employed in dicotyledonous crop plants such as soy, canola,cotton, sugar beet or potato.

A high expressing constitutive promoter as used in the application meansfor example a promoter which is functionally linked to a NEENA causingenhanced constitutive expression of the promoter in a plant or partthereof wherein the accumulation of RNA or rate of synthesis of RNAderived from the nucleic acid molecule under the control of therespective promoter functionally linked to a NEENA is higher, preferablysignificantly higher than the expression caused by the same promoterlacking a NEENA of the invention. Preferably the amount of RNA of therespective nucleic acid and/or the rate of RNA synthesis and/or the RNAstability in a plant is increased 50% or more, for example 100% or more,preferably 200% or more, more preferably 5 fold or more, even morepreferably 10 fold or more, most preferably 20 fold or more for example50 fold compared to a control plant of same age grown under the sameconditions comprising the same constitutive promoter the latter notbeing functionally linked to a NEENA of the invention.

When used herein, significantly higher refers to statisticalsignificance the skilled person is aware how to determine, for exampleby applying statistical tests such as the t-test to the respective datasets.

Methods for detecting expression conferred by a promoter are known inthe art. For example, the promoter may be functionally linked to amarker gene such as GUS, GFP or luciferase and the activity of therespective protein encoded by the respective marker gene may bedetermined in the plant or part thereof. As a representative example,the method for detecting luciferase is described in detail below. Othermethods are for example measuring the steady state level or synthesisrate of RNA of the nucleic acid molecule controlled by the promoter bymethods known in the art, for example Northern blot analysis, qPCR,run-on assays or other methods described in the art.

A skilled person is aware of various methods for functionally linkingtwo or more nucleic acid molecules. Such methods may encompassrestriction/ligation, ligase independent cloning, recombineering,recombination or synthesis. Other methods may be employed tofunctionally link two or more nucleic acid molecules.

A further embodiment of the present invention is a method for producinga plant or part thereof with, compared to a respective control plant orpart thereof, enhanced constitutive expression of one or more nucleicacid molecule comprising the steps of introducing into the plant or partthereof one or more NEENA comprising a nucleic acid molecule as definedabove under i) to vi) and functionally linking said one or more NEENA toa promoter, preferably a constitutive promoter and to a nucleic acidmolecule being under the control of said promoter, preferablyconstitutive promoter, wherein the NEENA is heterologous to said nucleicacid molecule.

The NEENA may be heterologous to the nucleic acid molecule which isunder the control of said promoter to which the NEENA is functionallylinked or it may be heterologous to both the promoter and the nucleicacid molecule under the control of said promoter.

The term “heterologous” with respect to a nucleic acid molecule or DNArefers to a nucleic acid molecule which is operably linked to, or ismanipulated to become operably linked to, a second nucleic acid moleculeto which it is not operably linked in nature, or to which it is operablylinked at a different location in nature. For example, a NEENA of theinvention is in its natural environment functionally linked to itsnative promoter, whereas in the present invention it is linked toanother promoter which might be derived from the same organism, adifferent organism or might be a synthetic promoter such as theSUPER-promoter. It may also mean that the NEENA of the present inventionis linked to its native promoter but the nucleic acid molecule undercontrol of said promoter is heterologous to the promoter comprising itsnative NEENA. It is in addition to be understood that the promoterand/or the nucleic acid molecule under the control of said promoterfunctionally linked to a NEENA of the invention are heterologous to saidNEENA as their sequence has been manipulated by for example mutationsuch as insertions, deletions and the forth so that the natural sequenceof the promoter and/or the nucleic acid molecule under control of saidpromoter is modified and therefore have become heterologous to a NEENAof the invention. It may also be understood that the NEENA isheterologous to the nucleic acid to which it is functionally linked whenthe NEENA is functionally linked to its native promoter wherein theposition of the NEENA in relation to said promoter is changed so thatthe promoter shows higher expression after such manipulation.

A plant exhibiting enhanced constitutive expression of a nucleic acidmolecule as meant herein means a plant having a higher, preferablystatistically significant higher constitutive expression of a nucleicacid molecule compared to a control plant grown under the sameconditions without the respective NEENA functionally linked to therespective nucleic acid molecule. Such control plant may be a wild-typeplant or a transgenic plant comprising the same promoter controlling thesame gene as in the plant of the invention wherein the promoter is notlinked to a NEENA of the invention.

Producing a plant as used herein comprises methods for stabletransformation such as introducing a recombinant DNA construct into aplant or part thereof by means of Agrobacterium mediated transformation,protoplast transformation, particle bombardment or the like andoptionally subsequent regeneration of a transgenic plant. It alsocomprises methods for transient transformation of a plant or partthereof such as viral infection or Agrobacterium infiltration. A skilledperson is aware of further methods for stable and/or transienttransformation of a plant or part thereof. Approaches such as breedingmethods or protoplast fusion might also be employed for production of aplant of the invention and are covered herewith.

The method of the invention may be applied to any plant, for examplegymnosperm or angiosperm, preferably angiosperm, for exampledicotyledonous or monocotyledonous plants, preferably dicotyledonousplants. Preferred monocotyledonous plants are for example corn, wheat,rice, barley, sorghum, musa, sugarcane, miscanthus and brachypodium,especially preferred monocotyledonous plants are corn, wheat and rice.Preferred dicotyledonous plants are for example soy, rape seed, canola,linseed, cotton, potato, sugar beet, tagetes and Arabidopsis, especiallypreferred dicotyledonous plants are soy, rape seed, canola and potato

In one embodiment of the invention, the methods as defined above arecomprising the steps of

a) introducing one or more NEENA comprising a nucleic acid molecule asdefined above in i) to vi) into a plant or part thereof and

b) integrating said one or more NEENA into the genome of said plant orpart thereof whereby said one or more NEENA is functionally linked to anendogenous preferably constitutively expressed nucleic acid heterologousto said one or more NEENA and optionally

c) regenerating a plant or part thereof comprising said one or moreNEENA from said transformed cell.

The NEENA may be heterologous to the nucleic acid molecule which isunder the control of said promoter to which the NEENA is functionallylinked or it may be heterologous to both the promoter and the nucleicacid molecule under the control of said promoter.

The one or more NEENA molecule may be introduced into the plant or partthereof by means of particle bombardment, protoplast electroporation,virus infection, Agrobacterium mediated transformation or any otherapproach known in the art. The NEENA molecule may be introducedintegrated for example into a plasmid or viral DNA or viral RNA. TheNEENA molecule may also be comprised on a BAC, YAC or artificialchromosome prior to introduction into the plant or part of the plant. Itmay be also introduced as a linear nucleic acid molecule comprising theNEENA sequence wherein additional sequences may be present adjacent tothe NEENA sequence on the nucleic acid molecule. These sequencesneighboring the NEENA sequence may be from about 20 bp, for example 20bp to several hundred base pairs, for example 100 bp or more and mayfacilitate integration into the genome for example by homologousrecombination. Any other method for genome integration may be employed,be it targeted integration approaches, such as homologous recombinationor random integration approaches, such as illegitimate recombination.

The endogenous preferably constitutively expressed nucleic acid to whichthe NEENA molecule may be functionally linked may be any nucleic acid,preferably any constitutively expressed nucleic acid molecule. Thenucleic acid molecule may be a protein coding nucleic acid molecule or anon coding molecule such as antisense RNA, rRNA, tRNA, miRNA, tasiRNA,siRNA, dsRNA, snRNA, snoRNA or any other noncoding RNA known in the art.The skilled person is aware of methods for identifying constitutivelyexpressed nucleic acid molecules to which the method of the inventionmay preferably be applied for example by microarray chip hybridization,qPCR, Northern blot analysis, next generation sequencing etc.

A further way to perform the methods of the invention may be to

a) provide an expression construct comprising one or more NEENAcomprising a nucleic acid molecule as defined above in i) to vi)functionally linked to a promoter, preferably a constitutive promoter asdefined above and to one or more nucleic acid molecule the latter beingheterologous to said one or more NEENA and which is under the control ofsaid promoter, preferably constitutive promoter and

b) integrate said expression construct comprising said one or more NEENAinto the genome of said plant or part thereof and optionally

c) regenerate a plant or part thereof comprising said one or moreexpression construct from said transformed plant or part thereof.

The NEENA may be heterologous to the nucleic acid molecule which isunder the control of said promoter to which the NEENA is functionallylinked or it may be heterologous to both the promoter and the nucleicacid molecule under the control of said promoter.

The expression construct may be integrated into the genome of therespective plant with any method known in the art. The integration maybe random using methods such as particle bombardment or Agrobacteriummediated transformation. In a preferred embodiment, the integration isvia targeted integration for example by homologous recombination. Thelatter method would allow integrating the expression constructcomprising a high expression promoter functionally linked to a NEENAinto a favorable genome region. Favorable genome regions are for examplegenome regions known to comprise genes that are highly expressed forexample in seeds and hence may increase expression derived from saidexpression construct compared to a genome region which shows notranscriptional activity.

In another preferred embodiment said one or more NEENA is functionallylinked to a promoter, preferably constitutive promoter close to thetranscription start site of said heterologous nucleic acid molecule.

Close to the transcription start site as meant herein comprisesfunctionally linking one or more NEENA to a promoter, preferably aconstitutive promoter 2500 bp or less, preferentially 2000 bp or less,more preferred 1500 bp or less, even more preferred 1000 bp or less andmost preferred 500 bp or less away from the transcription start site ofsaid heterologous nucleic acid molecule. It is to be understood that theNEENA may be integrated upstream or downstream in the respectivedistance from the transcription start site of the respective promoter.Hence, the one or more NEENA must not necessarily be included in thetranscript of the respective heterologous nucleic acid under control ofthe preferably constitutive promoter the one or more NEENA isfunctionally linked to. Preferentially the one or more NEENA isintegrated downstream of the transcription start site of the respectivepromoter, preferably constitutive promoter. The integration sitedownstream of the transcription start site may be in the 5′ UTR, the 3′UTR, an exon or intron or it may replace an intron or partially orcompletely the 5′ UTR or 3′ UTR of the heterologous nucleic acid underthe control of the preferably constitutive promoter. Preferentially theone or more NEENA is integrated in the 5′ UTR or an intron or the NEENAis replacing an intron or a part or the complete 5′UTR, mostpreferentially it is integrated in the 5′UTR of the respectiveheterologous nucleic acid.

A further embodiment of the invention comprises a recombinant expressionconstruct comprising one or more NEENA comprising a nucleic acidmolecule as defined above in i) to vi).

The recombinant expression construct may further comprise one or morepromoter, preferably constitutive promoter to which the one or moreNEENA is functionally linked and optionally one or more expressednucleic acid molecule the latter being heterologous to said one or moreNEENA.

The NEENA may be heterologous to the nucleic acid molecule which isunder the control of said promoter to which the NEENA is functionallylinked or it may be heterologous to both the promoter and the nucleicacid molecule under the control of said promoter.

The expression construct may comprise one ore more, for example two ormore, for example 5 or more, such as 10 or more combinations ofpromoters, preferably constitutive promoters functionally linked to aNEENA and a nucleic acid molecule to be expressed heterologous to therespective NEENA. The expression construct may also comprise furtherpromoters not comprising a NEENA functionally linked to nucleic acidmolecules to be expressed homologous or heterologous to the respectivepromoter.

A recombinant expression vector comprising one or more recombinantexpression construct as defined above is another embodiment of theinvention. A multitude of expression vectors that may be used in thepresent invention are known to a skilled person. Methods for introducingsuch a vector comprising such an expression construct comprising forexample a promoter functionally linked to a NEENA and optionally otherelements such as a terminator into the genome of a plant and forrecovering transgenic plants from a transformed cell are also well knownin the art. Depending on the method used for the transformation of aplant or part thereof the entire vector might be integrated into thegenome of said plant or part thereof or certain components of the vectormight be integrated into the genome, such as, for example a T-DNA.

A transgenic plant or part thereof comprising one or more heterologousNEENA as defined above in i) to vi) is also enclosed in this invention.A NEENA is to be understood as being heterologous to the plant if it issynthetic, derived from another organism or the same organism but itsnatural genomic localization is rendered compared to a control plant,for example a wild type plant. It is to be understood, that a renderedgenomic localization means the NEENA is located on another chromosome oron the same chromosome but 10 kb or more, for example 10 kb, preferably5 kb or more, for example 5 kb, more preferably 1000 bp or more, forexample 1000 bp, even more preferably 500 bp or more, for example 500bp, especially preferably 100bp or more, for example 100 bp, mostpreferably 10 bp or more, for example 10 bp dislocated from its naturalgenomic localization, for example in a wild type plant.

A transgenic cell or transgenic plant or part thereof comprising arecombinant expression vector as defined above or a recombinantexpression construct as defined above is a further embodiment of theinvention. The transgenic cell, transgenic plant or part thereof may beselected from the group consisting of bacteria, fungi, yeasts or plant,insect or mammalian cells or plants. Preferably the transgenic cells arebacteria, fungi, yeasts or plant cells. Preferred bacteria areEnterobacteria such as E. coli and bacteria of the genus Agrobacteria,for example Agrobacterium tumefaciens and Agrobacterium rhizogenes.Preferred plants are monocotyledonous or dicotyledonous plants forexample monocotyledonous or dicotyledonous crop plants such as corn,soy, canola, cotton, potato, sugar beet, rice, wheat, sorghum, barley,miscanthus, musa, sugarcane and the like. Preferred crop plants arecorn, rice, wheat, soy, canola, cotton or potato. Especially preferreddicotyledonous crop plants are soy, canola, cotton or potato.

Especially preferred monocotyledonous crop plants are corn, wheat andrice.

A transgenic cell culture, transgenic seed, parts or propagationmaterial derived from a transgenic cell or plant or part thereof asdefined above comprising said heterologous NEENA as defined above in i)to vi) or said recombinant expression construct or said recombinantvector as defined above are other embodiments of the invention.

Transgenic parts or propagation material as meant herein comprise alltissues and organs, for example leaf, stem and fruit as well as materialthat is useful for propagation and/or regeneration of plants such ascuttings, scions, layers, branches or shoots comprising the respectiveNEENA, recombinant expression construct or recombinant vector.

A further embodiment of the invention is the use of the NEENA as definedabove in i) to vi) or the recombinant construct or recombinant vector asdefined above for enhancing expression in plants or parts thereof.

Hence the application at hand provides seed-specific and/orseed-preferential gene expression enhancing nucleic acid moleculescomprising one or more promoter, preferably seedspecific and/or seedpreferential promoter functionally linked to one ore more NEENA.Additionally use of such gene expression enhancing nucleic acidmolecules and expression constructs, expression vectors, transgenicplants or parts thereof and transgenic cells comprising such geneexpression enhancing nucleic acid molecules are provided.

A use of a transgenic cell culture, transgenic seed, parts orpropagation material derived from a transgenic cell or plant or partthereof as defined above for the production of foodstuffs, animal feeds,seeds, pharmaceuticals or fine chemicals is also enclosed in thisinvention.

DEFINITIONS

Abbreviations: NEENA—nucleic acid expression enhancing nucleic acid,GFP—green fluorescence protein, GUS—beta-Glucuronidase,BAP—6-benzylaminopurine; 2,4-D—2,4-dichlorophenoxyacetic acid;MS—Murashige and Skoog medium; NAA—1-naphtaleneacetic acid; MES,2-(N-morpholino-ethanesulfonic acid, IAA indole acetic acid; Kan:Kanamycin sulfate; GA3—Gibberellic acid; Timentin™: ticarcillindisodium/clavulanate potassium, microl:Microliter.

It is to be understood that this invention is not limited to theparticular methodology or protocols. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims. It must benoted that as used herein and in the appended claims, the singular forms“a,” “and,” and “the” include plural reference unless the contextclearly dictates otherwise. Thus, for example, reference to “a vector”is a reference to one or more vectors and includes equivalents thereofknown to those skilled in the art, and so forth. The term “about” isused herein to mean approximately, roughly, around, or in the region of.When the term “about” is used in conjunction with a numerical range, itmodifies that range by extending the boundaries above and below thenumerical values set forth. In general, the term “about” is used hereinto modify a numerical value above and below the stated value by avariance of 20 percent, preferably 10 percent up or down (higher orlower). As used herein, the word “or” means any one member of aparticular list and also includes any combination of members of thatlist. The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of one or more stated features,integers, components, or steps, but they do not preclude the presence oraddition of one or more other features, integers, components, steps, orgroups thereof. For clarity, certain terms used in the specification aredefined and used as follows:

Antiparallel: “Antiparallel” refers herein to two nucleotide sequencespaired through hydrogen bonds between complementary base residues withphosphodiester bonds running in the 5′-3′ direction in one nucleotidesequence and in the 3′-5′ direction in the other nucleotide sequence.

Antisense: The term “antisense” refers to a nucleotide sequence that isinverted relative to its normal orientation for transcription orfunction and so expresses an RNA transcript that is complementary to atarget gene mRNA molecule expressed within the host cell (e.g., it canhybridize to the target gene mRNA molecule or single stranded genomicDNA through Watson-Crick base pairing) or that is complementary to atarget DNA molecule such as, for example genomic DNA present in the hostcell.

Coding region: As used herein the term “coding region” when used inreference to a structural gene refers to the nucleotide sequences whichencode the amino acids found in the nascent polypeptide as a result oftranslation of a mRNA molecule. The coding region is bounded, ineukaryotes, on the 5′-side by the nucleotide triplet “ATG” which encodesthe initiator methionine and on the 3′-side by one of the three tripletswhich specify stop codons (i.e., TAA, TAG, TGA). In addition tocontaining introns, genomic forms of a gene may also include sequenceslocated on both the 5′- and 3′-end of the sequences which are present onthe RNA transcript. These sequences are referred to as “flanking”sequences or regions (these flanking sequences are located 5′ or 3′ tothe non-translated sequences present on the mRNA transcript). The5′-flanking region may contain regulatory sequences such as promotersand enhancers which control or influence the transcription of the gene.The 3′-flanking region may contain sequences which direct thetermination of transcription, post-transcriptional cleavage andpolyadenylation.

Complementary: “Complementary” or “complementarity” refers to twonucleotide sequences which comprise antiparallel nucleotide sequencescapable of pairing with one another (by the base-pairing rules) uponformation of hydrogen bonds between the complementary base residues inthe antiparallel nucleotide sequences. For example, the sequence5′-AGT-3′ is complementary to the sequence 5′-ACT-3′. Complementaritycan be “partial” or “total.” “Partial” complementarity is where one ormore nucleic acid bases are not matched according to the base pairingrules. “Total” or “complete” complementarity between nucleic acidmolecules is where each and every nucleic acid base is matched withanother base under the base pairing rules. The degree of complementaritybetween nucleic acid molecule strands has significant effects on theefficiency and strength of hybridization between nucleic acid moleculestrands. A “complement” of a nucleic acid sequence as used herein refersto a nucleotide sequence whose nucleic acid molecules show totalcomplementarity to the nucleis acid molecules of the nucleic acidsequence.

Double-stranded RNA: A “double-stranded RNA” molecule or “dsRNA”molecule comprises a sense RNA fragment of a nucleotide sequence and anantisense RNA fragment of the nucleotide sequence, which both comprisenucleotide sequences complementary to one another, thereby allowing thesense and antisense RNA fragments to pair and form a double-stranded RNAmolecule.

Endogenous: An “endogenous” nucleotide sequence refers to a nucleotidesequence, which is present in the genome of the untransformed plantcell.

Enhanced expression: “enhance” or “increase” the expression of a nucleicacid molecule in a plant cell are used equivalently herein and mean thatthe level of expression of the nucleic acid molecule in a plant, part ofa plant or plant cell after applying a method of the present inventionis higher than its expression in the plant, part of the plant or plantcell before applying the method, or compared to a reference plantlacking a recombinant nucleic acid molecule of the invention. Forexample, the reference plant is comprising the same construct which isonly lacking the respective NEENA. The term “enhanced” or “increased” asused herein are synonymous and means herein higher, preferablysignificantly higher expression of the nucleic acid molecule to beexpressed. As used herein, an “enhancement” or “increase” of the levelof an agent such as a protein, mRNA or RNA means that the level isincreased relative to a substantially identical plant, part of a plantor plant cell grown under substantially identical conditions, lacking arecombinant nucleic acid molecule of the invention, for example lackingthe NEENA molecule, the recombinant construct or recombinant vector ofthe invetion. As used herein, “enhancement” or “increase” of the levelof an agent, such as for example a preRNA, mRNA, rRNA, tRNA, snoRNA,snRNA expressed by the target gene and/or of the protein product encodedby it, means that the level is increased 50% or more, for example 100%or more, preferably 200% or more, more preferably 5 fold or more, evenmore preferably 10 fold or more, most preferably 20 fold or more forexample 50 fold relative to a cell or organism lacking a recombinantnucleic acid molecule of the invention. The enhancement or increase canbe determined by methods with which the skilled worker is familiar.Thus, the enhancement or increase of the nucleic acid or proteinquantity can be determined for example by an immunological detection ofthe protein. Moreover, techniques such as protein assay, fluorescence,Northern hybridization, nuclease protection assay, reverse transcription(quantitative RT-PCR), ELISA (enzyme-linked immunosorbent assay),Western blotting, radioimmunoassay (RIA) or other immunoassays andfluorescence-activated cell analysis (FACS) can be employed to measure aspecific protein or RNA in a plant or plant cell. Depending on the typeof the induced protein product, its activity or the effect on thephenotype of the organism or the cell may also be determined. Methodsfor determining the protein quantity are known to the skilled worker.Examples, which may be mentioned, are: the micro-Biuret method (Goa J(1953) Scand J Clin Lab Invest 5:218-222), the Folin-Ciocalteau method(Lowry O H et al. (1951) J Biol Chem 193:265-275) or measuring theabsorption of CBB G-250 (Bradford M M (1976) Analyt Biochem 72:248-254).As one example for quantifying the activity of a protein, the detectionof luciferase activity is described in the Examples below.

Expression: “Expression” refers to the biosynthesis of a gene product,preferably to the transcription and/or translation of a nucleotidesequence, for example an endogenous gene or a heterologous gene, in acell. For example, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and—optionally—thesubsequent translation of mRNA into one or more polypeptides. In othercases, expression may refer only to the transcription of the DNAharboring an RNA molecule.

Expression construct: “Expression construct” as used herein mean a DNAsequence capable of directing expression of a particular nucleotidesequence in an appropriate part of a plant or plant cell, comprising apromoter functional in said part of a plant or plant cell into which itwill be introduced, operatively linked to the nucleotide sequence ofinterest which is—optionally—operatively linked to termination signals.If translation is required, it also typically comprises sequencesrequired for proper translation of the nucleotide sequence. The codingregion may code for a protein of interest but may also code for afunctional RNA of interest, for example RNAa, siRNA, snoRNA, snRNA,microRNA, to-siRNA or any other noncoding regulatory RNA, in the senseor antisense direction. The expression construct comprising thenucleotide sequence of interest may be chimeric, meaning that one ormore of its components is heterologous with respect to one or more ofits other components. The expression construct may also be one, which isnaturally occurring but has been obtained in a recombinant form usefulfor heterologous expression. Typically, however, the expressionconstruct is heterologous with respect to the host, i.e., the particularDNA sequence of the expression construct does not occur naturally in thehost cell and must have been introduced into the host cell or anancestor of the host cell by a transformation event. The expression ofthe nucleotide sequence in the expression construct may be under thecontrol of a constitutive promoter or of an inducible promoter, whichinitiates transcription only when the host cell is exposed to someparticular external stimulus. In the case of a plant, the promoter canalso be specific to a particular tissue or organ or stage ofdevelopment.

Foreign: The term “foreign” refers to any nucleic acid molecule (e.g.,gene sequence) which is introduced into the genome of a cell byexperimental manipulations and may include sequences found in that cellso long as the introduced sequence contains some modification (e.g., apoint mutation, the presence of a selectable marker gene, etc.) and istherefore distinct relative to the naturally-occurring sequence.

Functional linkage: The term “functional linkage” or “functionallylinked” is to be understood as meaning, for example, the sequentialarrangement of a regulatory element (e.g. a promoter) with a nucleicacid sequence to be expressed and, if appropriate, further regulatoryelements (such as e.g., a terminator or a NEENA) in such a way that eachof the regulatory elements can fulfill its intended function to allow,modify, facilitate or otherwise influence expression of said nucleicacid sequence. As a synonym the wording “operable linkage” or “operablylinked” may be used. The expression may result depending on thearrangement of the nucleic acid sequences in relation to sense orantisense RNA. To this end, direct linkage in the chemical sense is notnecessarily required. Genetic control sequences such as, for example,enhancer sequences, can also exert their function on the target sequencefrom positions which are further away, or indeed from other DNAmolecules. Preferred arrangements are those in which the nucleic acidsequence to be expressed recombinantly is positioned behind the sequenceacting as promoter, so that the two sequences are linked covalently toeach other. The distance between the promoter sequence and the nucleicacid sequence to be expressed recombinantly is preferably less than 200base pairs, especially preferably less than 100 base pairs, veryespecially preferably less than 50 base pairs. In a preferredembodiment, the nucleic acid sequence to be transcribed is locatedbehind the promoter in such a way that the transcription start isidentical with the desired beginning of the chimeric RNA of theinvention. Functional linkage, and an expression construct, can begenerated by means of customary recombination and cloning techniques asdescribed (e.g., in Maniatis T, Fritsch E F and Sambrook J (1989)Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Cold Spring Harbor (N.Y.); Silhavy et al. (1984) Experimentswith Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor(N.Y.); Ausubel et al. (1987) Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley Interscience; Gelvin et al. (Eds)(1990) Plant Molecular Biology Manual; Kluwer Academic Publisher,Dordrecht, The Netherlands). However, further sequences, which, forexample, act as a linker with specific cleavage sites for restrictionenzymes, or as a signal peptide, may also be positioned between the twosequences. The insertion of sequences may also lead to the expression offusion proteins. Preferably, the expression construct, consisting of alinkage of a regulatory region for example a promoter and nucleic acidsequence to be expressed, can exist in a vector-integrated form and beinserted into a plant genome, for example by transformation.

Gene: The term “gene” refers to a region operably joined to appropriateregulatory sequences capable of regulating the expression of the geneproduct (e.g., a polypeptide or a functional RNA) in some manner. A geneincludes untranslated regulatory regions of DNA (e.g., promoters,enhancers, repressors, etc.) preceding (up-stream) and following(downstream) the coding region (open reading frame, ORF) as well as,where applicable, intervening sequences (i.e., introns) betweenindividual coding regions (i.e., exons). The term “structural gene” asused herein is intended to mean a DNA sequence that is transcribed intomRNA which is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

Genome and genomic DNA: The terms “genome” or “genomic DNA” is referringto the heritable genetic information of a host organism. Said genomicDNA comprises the DNA of the nucleus (also referred to as chromosomalDNA) but also the DNA of the plastids (e.g., chloroplasts) and othercellular organelles (e.g., mitochondria). Preferably the terms genome orgenomic DNA is referring to the chromosomal DNA of the nucleus.

Heterologous: The term “heterologous” with respect to a nucleic acidmolecule or DNA refers to a nucleic acid molecule which is operablylinked to, or is manipulated to become operably linked to, a secondnucleic acid molecule to which it is not operably linked in nature, orto which it is operably linked at a different location in nature. Aheterologous expression construct comprising a nucleic acid molecule andone or more regulatory nucleic acid molecule (such as a promoter or atranscription termination signal) linked thereto for example is aconstructs originating by experimental manipulations in which either a)said nucleic acid molecule, or b) said regulatory nucleic acid moleculeor c) both (i.e. (a) and (b)) is not located in its natural (native)genetic environment or has been modified by experimental manipulations,an example of a modification being a substitution, addition, deletion,inversion or insertion of one or more nucleotide residues. Naturalgenetic environment refers to the natural chromosomal locus in theorganism of origin, or to the presence in a genomic library. In the caseof a genomic library, the natural genetic environment of the sequence ofthe nucleic acid molecule is preferably retained, at least in part. Theenvironment flanks the nucleic acid sequence at least at one side andhas a sequence of at least 50 bp, preferably at least 500 bp, especiallypreferably at least 1,000 bp, very especially preferably at least 5,000bp, in length. A naturally occurring expression construct—for examplethe naturally occurring combination of a promoter with the correspondinggene—becomes a transgenic expression construct when it is modified bynon-natural, synthetic “artificial” methods such as, for example,mutagenization. Such methods have been described (U.S. Pat. No.5,565,350; WO 00/15815). For example a protein encoding nucleic acidmolecule operably linked to a promoter, which is not the native promoterof this molecule, is considered to be heterologous with respect to thepromoter. Preferably, heterologous DNA is not endogenous to or notnaturally associated with the cell into which it is introduced, but hasbeen obtained from another cell or has been synthesized. HeterologousDNA also includes an endogenous DNA sequence, which contains somemodification, non-naturally occurring, multiple copies of an endogenousDNA sequence, or a DNA sequence which is not naturally associated withanother DNA sequence physically linked thereto. Generally, although notnecessarily, heterologous DNA encodes RNA or proteins that are notnormally produced by the cell into which it is expressed.

High expression constitutive promoter: A “high expression constitutivepromoter” as used herein means a promoter causing constitutiveexpression in a plant or part thereof wherein the accumulation or rateof synthesis of RNA or stability of RNA derived from the nucleic acidmolecule under the control of the respective promoter is higher,preferably significantly higher than the expression caused by thepromoter lacking the NEENA of the invention. Preferably the amount ofRNA and/or the rate of RNA synthesis and/or stability of RNA isincreased 50% or more, for example 100% or more, preferably 200% ormore, more preferably 5 fold or more, even more preferably 10 fold ormore, most preferably 20 fold or more for example 50 fold relative to aconstitutive promoter lacking a NEENA of the invention.

Hybridization: The term “hybridization” as used herein includes “anyprocess by which a strand of nucleic acid molecule joins with acomplementary strand through base pairing.” (J. Coombs (1994) Dictionaryof Biotechnology, Stockton Press, New York). Hybridization and thestrength of hybridization (i.e., the strength of the association betweenthe nucleic acid molecules) is impacted by such factors as the degree ofcomplementarity between the nucleic acid molecules, stringency of theconditions involved, the Tm of the formed hybrid, and the G:C ratiowithin the nucleic acid molecules. As used herein, the term “Tm” is usedin reference to the “melting temperature.” The melting temperature isthe temperature at which a population of double-stranded nucleic acidmolecules becomes half dissociated into single strands. The equation forcalculating the Tm of nucleic acid molecules is well known in the art.As indicated by standard references, a simple estimate of the Tm valuemay be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleicacid molecule is in aqueous solution at 1 M NaCl [see e.g., Anderson andYoung, Quantitative Filter Hybridization, in Nucleic Acid Hybridization(1985)]. Other references include more sophisticated computations, whichtake structural as well as sequence characteristics into account for thecalculation of Tm. Stringent conditions, are known to those skilled inthe art and can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

“Identity”: “Identity” when used in respect to the comparison of two ormore nucleic acid or amino acid molecules means that the sequences ofsaid molecules share a certain degree of sequence similarity, thesequences being partially identical.

To determine the percentage identity (homology is herein usedinterchangeably) of two amino acid sequences or of two nucleic acidmolecules, the sequences are written one underneath the other for anoptimal comparison (for example gaps may be inserted into the sequenceof a protein or of a nucleic acid in order to generate an optimalalignment with the other protein or the other nucleic acid).

The amino acid residues or nucleic acid molecules at the correspondingamino acid positions or nucleotide positions are then compared. If aposition in one sequence is occupied by the same amino acid residue orthe same nucleic acid molecule as the corresponding position in theother sequence, the molecules are homologous at this position (i.e.amino acid or nucleic acid “homology” as used in the present contextcorresponds to amino acid or nucleic acid “identity”. The percentageidentity between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e. % homology=number ofidentical positions/total number of positions×100). The terms “homology”and “identity” are thus to be considered as synonyms.

For the determination of the percentage identity of two or more aminoacids or of two or more nucleotide sequences several computer softwareprograms have been developed. The identity of two or more sequences canbe calculated with for example the software fasta, which presently hasbeen used in the version fasta 3 (W. R. Pearson and D. J. Lipman, PNAS85, 2444(1988); W. R. Pearson, Methods in Enzymology 183, 63 (1990); W.R. Pearson and D. J. Lipman, PNAS 85, 2444 (1988); W. R. Pearson,Enzymology 183, 63 (1990)). Another useful program for the calculationof identities of different sequences is the standard blast program,which is included in the Biomax pedant software (Biomax, Munich, FederalRepublic of Germany). This leads unfortunately sometimes to suboptimalresults since blast does not always include complete sequences of thesubject and the query. Nevertheless as this program is very efficient itcan be used for the comparison of a huge number of sequences. Thefollowing settings are typically used for such a comparisons ofsequences:

-p Program Name [String]; -d Database [String]; default=nr; -i QueryFile [File In]; default=stdin; -e Expectation value (E) [Real];default=10.0; -m alignment view options: 0=pairwise; 1=query-anchoredshowing identities; 2=query-anchored no identities; 3=flatquery-anchored, show identities; 4=flat query-anchored, no identities;5=query-anchored no identities and blunt ends; 6=flat query-anchored, noidentities and blunt ends; 7=XML Blast output; 8=tabular; 9 tabular withcomment lines [Integer]; default=0; -o BLAST report Output File [FileOut] Optional; default=stdout; -F Filter query sequence (DUST withblastn, SEG with others) [String]; default=T; -G Cost to open a gap(zero invokes default behavior) [Integer]; default=0; -E Cost to extenda gap (zero invokes default behavior) [Integer]; default=0; -X X dropoffvalue for gapped alignment (in bits) (zero invokes default behavior);blastn 30, megablast 20, tblastx 0, all others 15 [Integer]; default=0;-I Show GI's in deflines [T/F]; default=F; -q Penalty for a nucleotidemismatch (blastn only) [Integer]; default=-3; -r Reward for a nucleotidematch (blastn only) [Integer]; default=1; -v Number of databasesequences to show one-line descriptions for (V) [Integer]; default=500;-b Number of database sequence to show alignments for (B) [Integer];default=250; -f Threshold for extending hits, default if zero; blastp11, blastn 0, blastx 12, tblastn 13; tblastx 13, megablast 0 [Integer];default=0; -g Perfom gapped alignment (not available with tblastx)[T/F]; default=T; -Q Query Genetic code to use [Integer]; default=1; -DDB Genetic code (for tblast[nx] only) [Integer]; default=1; -a Number ofprocessors to use [Integer]; default=1; -O SeqAlign file [File Out]Optional; -J Believe the query defline [T/F]; default=F; -M Matrix[String]; default=BLOSUM62; -W Word size, default if zero (blastn 11,megablast 28, all others 3) [Integer]; default=0; -z Effective length ofthe database (use zero for the real size) [Real]; default=0; -K Numberof best hits from a region to keep (off by default, if used a value of100 is recommended) [Integer]; default=0; -P 0 for multiple hit, 1 forsingle hit [Integer]; default=0; -Y Effective length of the search space(use zero for the real size) [Real]; default=0; -S Query strands tosearch against database (for blast[nx], and tblastx); 3 is both, 1 istop, 2 is bottom [Integer]; default=3; -T Produce HTML output [T/F];default=F; -I Restrict search of database to list of GI's [String]Optional; -U Use lower case filtering of FASTA sequence [T/F] Optional;default=F; -y X dropoff value for ungapped extensions in bits (0.0invokes default behavior); blastn 20, megablast 10, all others 7[Real];default=0.0; -Z X dropoff value for final gapped alignment in bits (0.0invokes default behavior); blastn/megablast 50, tblastx 0, all others 25[Integer]; default=0; -R PSI-TBLASTN checkpoint file [File In] Optional;-n MegaBlast search [T/F]; default=F; -L Location on query sequence[String] Optional; -A Multiple Hits window size, default if zero(blastn/megablast 0, all others 40 [Integer]; default=0; -w Frame shiftpenalty (OOF algorithm for blastx) [Integer]; default=0; -t Length ofthe largest intron allowed in tblastn for linking HSPs (0 disableslinking) [Integer]; default=0.

Results of high quality are reached by using the algorithm of Needlemanand Wunsch or Smith and Waterman. Therefore programs based on saidalgorithms are preferred. Advantageously the comparisons of sequencescan be done with the program PileUp (J. Mol. Evolution., 25, 351 (1987),Higgins et al., CABIOS 5, 151 (1989)) or preferably with the programs“Gap” and “Needle”, which are both based on the algorithms of Needlemanand Wunsch (J. Mol. Biol. 48; 443 (1970)), and “BestFit”, which is basedon the algorithm of Smith and Waterman (Adv. Appl. Math. 2; 482 (1981)).“Gap” and “BestFit” are part of the GCG software-package (GeneticsComputer Group, 575 Science Drive, Madison, Wis., USA 53711 (1991);Altschul et al., (Nucleic Acids Res. 25, 3389 (1997)), “Needle” is partof the The European Molecular Biology Open Software Suite (EMBOSS)(Trends in Genetics 16 (6), 276 (2000)). Therefore preferably thecalculations to determine the percentages of sequence identity are donewith the programs “Gap” or “Needle” over the whole range of thesequences. The following standard adjustments for the comparison ofnucleic acid sequences were used for “Needle”: matrix: EDNAFULL,Gap_penalty: 10.0, Extend_penalty: 0.5. The following standardadjustments for the comparison of nucleic acid sequences were used for“Gap”: gap weight: 50, length weight: 3, average match: 10.000, averagemismatch: 0.000.

For example a sequence, which is said to have 80% identity with sequenceSEQ ID NO: 1 at the nucleic acid level is understood as meaning asequence which, upon comparison with the sequence represented by SEQ IDNO: 1 by the above program “Needle” with the above parameter set, has a80% identity. Preferably the identity is calculated on the completelength of the query sequence, for example SEQ ID NO:1.

Intron: refers to sections of DNA (intervening sequences) within a genethat do not encode part of the protein that the gene produces, and thatis spliced out of the mRNA that is transcribed from the gene before itis exported from the cell nucleus. Intron sequence refers to the nucleicacid sequence of an intron. Thus, introns are those regions of DNAsequences that are transcribed along with the coding sequence (exons)but are removed during the formation of mature mRNA. Introns can bepositioned within the actual coding region or in either the 5′ or 3′untranslated leaders of the pre-mRNA (unspliced mRNA). Introns in theprimary transcript are excised and the coding sequences aresimultaneously and precisely ligated to form the mature mRNA. Thejunctions of introns and exons form the splice site. The sequence of anintron begins with GU and ends with AG. Furthermore, in plants, twoexamples of AU-AC introns have been described: the fourteenth intron ofthe RecA-like protein gene and the seventh intron of the G5 gene fromArabidopsis thaliana are AT-AC introns. Pre-mRNAs containing intronshave three short sequences that are—beside other sequences—essential forthe intron to be accurately spliced. These sequences are the 5′splice-site, the 3′ splice-site, and the branchpoint. mRNA splicing isthe removal of intervening sequences (introns) present in primary mRNAtranscripts and joining or ligation of exon sequences. This is alsoknown as cis-splicing which joins two exons on the same RNA with theremoval of the intervening sequence (intron). The functional elements ofan intron is comprising sequences that are recognized and bound by thespecific protein components of the spliceosome (e.g. splicing consensussequences at the ends of introns). The interaction of the functionalelements with the spliceosome results in the removal of the intronsequence from the premature mRNA and the rejoining of the exonsequences. Introns have three short sequences that are essential-although not sufficient-for the intron to be accurately spliced. Thesesequences are the 5′ splice site, the 3′ splice site and the branchpoint. The branchpoint sequence is important in splicing and splice-siteselection in plants. The branchpoint sequence is usually located 10-60nucleotides upstream of the 3′ splice site.

Isogenic: organisms (e.g., plants), which are genetically identical,except that they may differ by the presence or absence of a heterologousDNA sequence.

Isolated: The term “isolated” as used herein means that a material hasbeen removed by the hand of man and exists apart from its original,native environment and is therefore not a product of nature. An isolatedmaterial or molecule (such as a DNA molecule or enzyme) may exist in apurified form or may exist in a non-native environment such as, forexample, in a transgenic host cell. For example, a naturally occurringpolynucleotide or polypeptide present in a living plant is not isolated,but the same polynucleotide or polypeptide, separated from some or allof the coexisting materials in the natural system, is isolated. Suchpolynucleotides can be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and would be isolated inthat such a vector or composition is not part of its originalenvironment. Preferably, the term “isolated” when used in relation to anucleic acid molecule, as in “an isolated nucleic acid sequence” refersto a nucleic acid sequence that is identified and separated from atleast one contaminant nucleic acid molecule with which it is ordinarilyassociated in its natural source. Isolated nucleic acid molecule isnucleic acid molecule present in a form or setting that is differentfrom that in which it is found in nature. In contrast, non-isolatednucleic acid molecules are nucleic acid molecules such as DNA and RNA,which are found in the state they exist in nature. For example, a givenDNA sequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs, which encode a multitude of proteins.However, an isolated nucleic acid sequence comprising for example SEQ IDNO: 1 includes, by way of example, such nucleic acid sequences in cellswhich ordinarily contain SEQ ID NO:1 where the nucleic acid sequence isin a chromosomal or extrachromosomal location different from that ofnatural cells, or is otherwise flanked by a different nucleic acidsequence than that found in nature. The isolated nucleic acid sequencemay be present in single-stranded or double-stranded form. When anisolated nucleic acid sequence is to be utilized to express a protein,the nucleic acid sequence will contain at a minimum at least a portionof the sense or coding strand (i.e., the nucleic acid sequence may besingle-stranded). Alternatively, it may contain both the sense andanti-sense strands (i.e., the nucleic acid sequence may bedouble-stranded).

Minimal Promoter: promoter elements, particularly a TATA element, thatare inactive or that have greatly reduced promoter activity in theabsence of upstream activation. In the presence of a suitabletranscription factor, the minimal promoter functions to permittranscription. NEENA: see “Nucleic acid expression enhancing nucleicacid”.

Non-coding: The term “non-coding” refers to sequences of nucleic acidmolecules that do not encode part or all of an expressed protein.Non-coding sequences include but are not limited to introns, enhancers,promoter regions, 3′ untranslated regions, and 5′ untranslated regions.

Nucleic acid expression enhancing nucleic acid (NEENA): The term“nucleic acid expression enhancing nucleic acid” refers to a sequenceand/or a nucleic acid molecule of a specific sequence having theintrinsic property to enhance expression of a nucleic acid under thecontrol of a promoter to which the NEENA is functionally linked. Unlikepromoter sequences, the NEENA as such is not able to drive expression.In order to fulfill the function of enhancing expression of a nucleicacid molecule functionally linked to the NEENA, the NEENA itself has tobe functionally linked to a promoter. In distinction to enhancersequences known in the art, the NEENA is acting in cis but not in transand has to be located close to the transcription start site of thenucleic acid to be expressed.

Nucleic acids and nucleotides: The terms “Nucleic Acids” and“Nucleotides” refer to naturally occurring or synthetic or artificialnucleic acid or nucleotides. The terms “nucleic acids” and “nucleotides”comprise deoxyribonucleotides or ribonucleotides or any nucleotideanalogue and polymers or hybrids thereof in either single- ordouble-stranded, sense or antisense form. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences, as well as the sequenceexplicitly indicated. The term “nucleic acid” is used inter-changeablyherein with “gene”, “cDNA, “mRNA”, “oligonucleotide,” and“polynucleotide”. Nucleotide analogues include nucleotides havingmodifications in the chemical structure of the base, sugar and/orphosphate, including, but not limited to, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atcytosine exocyclic amines, substitution of 5-bromo-uracil, and the like;and 2′-position sugar modifications, including but not limited to,sugar-modified ribonucleotides in which the 2′-OH is replaced by a groupselected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN. Shorthairpin RNAs (shRNAs) also can comprise non-natural elements such asnon-natural bases, e.g., ionosin and xanthine, non-natural sugars, e.g.,2′-methoxy ribose, or non-natural phosphodiester linkages, e.g.,methylphosphonates, phosphorothioates and peptides.

Nucleic acid sequence: The phrase “nucleic acid sequence” refers to asingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′-to the 3′-end. It includeschromosomal DNA, self-replicating plasmids, infectious polymers of DNAor RNA and DNA or RNA that performs a primarily structural role.“Nucleic acid sequence” also refers to a consecutive list ofabbreviations, letters, characters or words, which representnucleotides. In one embodiment, a nucleic acid can be a “probe” which isa relatively short nucleic acid, usually less than 100 nucleotides inlength. Often a nucleic acid probe is from about 50 nucleotides inlength to about 10 nucleotides in length. A “target region” of a nucleicacid is a portion of a nucleic acid that is identified to be ofinterest. A “coding region” of a nucleic acid is the portion of thenucleic acid, which is transcribed and translated in a sequence-specificmanner to produce into a particular polypeptide or protein when placedunder the control of appropriate regulatory sequences. The coding regionis said to encode such a polypeptide or protein.

Oligonucleotide: The term “oligonucleotide” refers to an oligomer orpolymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) ormimetics thereof, as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. An oligonucleotide preferablyincludes two or more nucleomonomers covalently coupled to each other bylinkages (e.g., phosphodiesters) or substitute linkages.

Overhang: An “overhang” is a relatively short single-stranded nucleotidesequence on the 5′- or 3′-hydroxyl end of a double-strandedoligonucleotide molecule (also referred to as an “extension,”“protruding end,” or “sticky end”).

Plant: is generally understood as meaning any eukaryotic single-ormulti-celled organism or a cell, tissue, organ, part or propagationmaterial (such as seeds or fruit) of same which is capable ofphotosynthesis. Included for the purpose of the invention are all generaand species of higher and lower plants of the Plant Kingdom. Annual,perennial, monocotyledonous and dicotyledonous plants are preferred. Theterm includes the mature plants, seed, shoots and seedlings and theirderived parts, propagation material (such as seeds or microspores),plant organs, tissue, protoplasts, callus and other cultures, forexample cell cultures, and any other type of plant cell grouping to givefunctional or structural units. Mature plants refer to plants at anydesired developmental stage beyond that of the seedling. Seedling refersto a young immature plant at an early developmental stage. Annual,biennial, monocotyledonous and dicotyledonous plants are preferred hostorganisms for the generation of transgenic plants. The expression ofgenes is furthermore advantageous in all ornamental plants, useful orornamental trees, flowers, cut flowers, shrubs or lawns. Plants whichmay be mentioned by way of example but not by limitation areangiosperms, bryophytes such as, for example, Hepaticae (liverworts) andMusci (mosses); Pteridophytes such as ferns, horsetail and club mosses;gymnosperms such as conifers, cycads, ginkgo and Gnetatae; algae such asChlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae,Bacillariophyceae (diatoms), and Euglenophyceae. Preferred are plantswhich are used for food or feed purpose such as the families of theLeguminosae such as pea, alfalfa and soya; Gramineae such as rice,maize, wheat, barley, sorghum, millet, rye, triticale, or oats; thefamily of the Umbelliferae, especially the genus Daucus, very especiallythe species carota (carrot) and Apium, very especially the speciesGraveolens dulce (celery) and many others; the family of the Solanaceae,especially the genus Lycopersicon, very especially the speciesesculentum (tomato) and the genus Solanum, very especially the speciestuberosum (potato) and melongena (egg plant), and many others (such astobacco); and the genus Capsicum, very especially the species annuum(peppers) and many others; the family of the Leguminosae, especially thegenus Glycine, very especially the species max (soybean), alfalfa, pea,lucerne, beans or peanut and many others; and the family of theCruciferae (Brassicacae), especially the genus Brassica, very especiallythe species napus (oil seed rape), campestris (beet), oleracea cv Tastie(cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor(broccoli); and of the genus Arabidopsis, very especially the speciesthaliana and many others; the family of the Compositae, especially thegenus Lactuca, very especially the species sativa (lettuce) and manyothers; the family of the Asteraceae such as sunflower, Tagetes, lettuceor Calendula and many other; the family of the Cucurbitaceae such asmelon, pumpkin/squash or zucchini, and linseed. Further preferred arecotton, sugar cane, hemp, flax, chillies, and the various tree, nut andwine species.

Polypeptide: The terms “polypeptide”, “peptide”, “oligopeptide”,“polypeptide”, “gene product”, “expression product” and “protein” areused interchangeably herein to refer to a polymer or oligomer ofconsecutive amino acid residues.

Pre-protein: Protein, which is normally targeted to a cellularorganelle, such as a chloroplast, and still comprising its transitpeptide.

Primary transcript: The term “primary transcript” as used herein refersto a premature RNA transcript of a gene. A “primary transcript” forexample still comprises introns and/or is not yet comprising a polyAtail or a cap structure and/or is missing other modifications necessaryfor its correct function as transcript such as for example trimming orediting.

Promoter: The terms “promoter”, or “promoter sequence” are equivalentsand as used herein, refer to a DNA sequence which when ligated to anucleotide sequence of interest is capable of controlling thetranscription of the nucleotide sequence of interest into RNA. Suchpromoters can for example be found in the following public databasesgrassius.org/grasspromdb.html,mendel.cs.rhul.ac.uk/mendel.php?topic=plantprom,ppdb.gene.nagoya-u.ac.jp/cgi-bin/index.cgi. Promoters listed there maybe addressed with the methods of the invention and are herewith includedby reference. A promoter is located 5′ (i.e., upstream), proximal to thetranscriptional start site of a nucleotide sequence of interest whosetranscription into mRNA it controls, and provides a site for specificbinding by RNA polymerase and other transcription factors for initiationof transcription. Said promoter comprises for example the at least 10kb, for example 5 kb or 2 kb proximal to the transcription start site.It may also comprise the at least 1500 bp proximal to thetranscriptional start site, preferably the at least 1000 bp, morepreferably the at least 500 bp, even more preferably the at least 400bp, the at least 300 bp, the at least 200 bp or the at least 100 bp. Ina further preferred embodiment, the promoter comprises the at least 50bp proximal to the transcription start site, for example, at least 25bp. The promoter does not comprise exon and/or intron regions or 5′untranslated regions. The promoter may for example be heterologous orhomologous to the respective plant. A polynucleotide sequence is“heterologous to” an organism or a second polynucleotide sequence if itoriginates from a foreign species, or, if from the same species, ismodified from its original form. For example, a promoter operably linkedto a heterologous coding sequence refers to a coding sequence from aspecies different from that from which the promoter was derived, or, iffrom the same species, a coding sequence which is not naturallyassociated with the promoter (e.g. a genetically engineered codingsequence or an allele from a different ecotype or variety). Suitablepromoters can be derived from genes of the host cells where expressionshould occur or from pathogens for this host cells (e.g., plants orplant pathogens like plant viruses). A plant specific promoter is apromoter suitable for regulating expression in a plant. It may bederived from a plant but also from plant pathogens or it might be asynthetic promoter designed by man. If a promoter is an induciblepromoter, then the rate of transcription increases in response to aninducing agent. Also, the promoter may be regulated in a tissue-specificor tissue preferred manner such that it is only or predominantly activein transcribing the associated coding region in a specific tissuetype(s) such as leaves, roots or meristem. The term “tissue specific” asit applies to a promoter refers to a promoter that is capable ofdirecting selective expression of a nucleotide sequence of interest to aspecific type of tissue (e.g., petals) in the relative absence ofexpression of the same nucleotide sequence of interest in a differenttype of tissue (e.g., roots). Tissue specificity of a promoter may beevaluated by, for example, operably linking a reporter gene to thepromoter sequence to generate a reporter construct, introducing thereporter construct into the genome of a plant such that the reporterconstruct is integrated into every tissue of the resulting transgenicplant, and detecting the expression of the reporter gene (e.g.,detecting mRNA, protein, or the activity of a protein encoded by thereporter gene) in different tissues of the transgenic plant. Thedetection of a greater level of expression of the reporter gene in oneor more tissues relative to the level of expression of the reporter genein other tissues shows that the promoter is specific for the tissues inwhich greater levels of expression are detected. The term “cell typespecific” as applied to a promoter refers to a promoter, which iscapable of directing selective expression of a nucleotide sequence ofinterest in a specific type of cell in the relative absence ofexpression of the same nucleotide sequence of interest in a differenttype of cell within the same tissue. The term “cell type specific” whenapplied to a promoter also means a promoter capable of promotingselective expression of a nucleotide sequence of interest in a regionwithin a single tissue. Cell type specificity of a promoter may beassessed using methods well known in the art, e.g., GUS activitystaining, GFP protein or immunohistochemical staining. The term“constitutive” when made in reference to a promoter or the expressionderived from a promoter means that the promoter is capable of directingtranscription of an operably linked nucleic acid molecule in the absenceof a stimulus (e.g., heat shock, chemicals, light, etc.) in the majorityof plant tissues and cells throughout substantially the entire lifespanof a plant or part of a plant. Typically, constitutive promoters arecapable of directing expression of a transgene in substantially any celland any tissue.

Promoter specificity: The term “specificity” when referring to apromoter means the pattern of expression conferred by the respectivepromoter. The specificity describes the tissues and/or developmentalstatus of a plant or part thereof, in which the promoter is conferringexpression of the nucleic acid molecule under the control of therespective promoter. Specificity of a promoter may also comprise theenvironmental conditions, under which the promoter may be activated ordown-regulated such as induction or repression by biological orenvironmental stresses such as cold, drought, wounding or infection.

Purified: As used herein, the term “purified” refers to molecules,either nucleic or amino acid sequences that are removed from theirnatural environment, isolated or separated. “Substantially purified”molecules are at least 60% free, preferably at least 75% free, and morepreferably at least 90% free from other components with which they arenaturally associated.

A purified nucleic acid sequence may be an isolated nucleic acidsequence.

Recombinant: The term “recombinant” with respect to nucleic acidmolecules refers to nucleic acid molecules produced by recombinant DNAtechniques. Recombinant nucleic acid molecules may also comprisemolecules, which as such does not exist in nature but are modified,changed, mutated or otherwise manipulated by man. Preferably, a“recombinant nucleic acid molecule” is a non-naturally occurring nucleicacid molecule that differs in sequence from a naturally occurringnucleic acid molecule by at least one nucleic acid. A “recombinantnucleic acid molecule” may also comprise a “recombinant construct” whichcomprises, preferably operably linked, a sequence of nucleic acidmolecules not naturally occurring in that order. Preferred methods forproducing said recombinant nucleic acid molecule may comprise cloningtechniques, directed or non-directed mutagenesis, synthesis orrecombination techniques.

Sense: The term “sense” is understood to mean a nucleic acid moleculehaving a sequence which is complementary or identical to a targetsequence, for example a sequence which binds to a protein transcriptionfactor and which is involved in the expression of a given gene.According to a preferred embodiment, the nucleic acid molecule comprisesa gene of interest and elements allowing the expression of the said geneof interest.

Significant increase or decrease: An increase or decrease, for examplein enzymatic activity or in gene expression, that is larger than themargin of error inherent in the measurement technique, preferably anincrease or decrease by about 2-fold or greater of the activity of thecontrol enzyme or expression in the control cell, more preferably anincrease or decrease by about 5-fold or greater, and most preferably anincrease or decrease by about 10-fold or greater.

Small nucleic acid molecules: “small nucleic acid molecules” areunderstood as molecules consisting of nucleic acids or derivativesthereof such as RNA or DNA. They may be doublestranded orsingle-stranded and are between about 15 and about 30 bp, for examplebetween 15 and 30 bp, more preferred between about 19 and about 26 bp,for example between 19 and 26 bp, even more preferred between about 20and about 25 bp for example between 20 and 25 bp. In a especiallypreferred embodiment the oligonucleotides are between about 21 and about24 bp, for example between 21 and 24 bp. In a most preferred embodiment,the small nucleic acid molecules are about 21 bp and about 24 bp, forexample 21 bp and 24 bp.

Substantially complementary: In its broadest sense, the term“substantially complementary”, when used herein with respect to anucleotide sequence in relation to a reference or target nucleotidesequence, means a nucleotide sequence having a percentage of identitybetween the substantially complementary nucleotide sequence and theexact complementary sequence of said reference or target nucleotidesequence of at least 60%, more desirably at least 70%, more desirably atleast 80% or 85%, preferably at least 90%, more preferably at least 93%,still more preferably at least 95% or 96%, yet still more preferably atleast 97% or 98%, yet still more preferably at least 99% or mostpreferably 100% (the later being equivalent to the term “identical” inthis context). Preferably identity is assessed over a length of at least19 nucleotides, preferably at least 50 nucleotides, more preferably theentire length of the nucleic acid sequence to said reference sequence(if not specified otherwise below). Sequence comparisons are carried outusing default GAP analysis with the University of Wisconsin GCG, SEQWEBapplication of GAP, based on the algorithm of Needleman and Wunsch(Needleman and Wunsch (1970) J Mol. Biol. 48: 443-453; as definedabove). A nucleotide sequence “substantially complementary ” to areference nucleotide sequence hybridizes to the reference nucleotidesequence under low stringency conditions, preferably medium stringencyconditions, most preferably high stringency conditions (as definedabove).

Transgene: The term “transgene” as used herein refers to any nucleicacid sequence, which is introduced into the genome of a cell byexperimental manipulations. A transgene may be an “endogenous DNAsequence,” or a “heterologous DNA sequence” (i.e., “foreign DNA”). Theterm “endogenous DNA sequence” refers to a nucleotide sequence, which isnaturally found in the cell into which it is introduced so long as itdoes not contain some modification (e.g., a point mutation, the presenceof a selectable marker gene, etc.) relative to the naturally-occurringsequence.

Transgenic: The term transgenic when referring to an organism meanstransformed, preferably stably transformed, with a recombinant DNAmolecule that preferably comprises a suitable promoter operativelylinked to a DNA sequence of interest.

Vector: As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid molecule to whichit has been linked. One type of vector is a genomic integrated vector,or “integrated vector”, which can become integrated into the chromosomalDNA of the host cell. Another type of vector is an episomal vector,i.e., a nucleic acid molecule capable of extra-chromosomal replication.Vectors capable of directing the expression of genes to which they areoperatively linked are referred to herein as “expression vectors”. Inthe present specification, “plasmid” and “vector” are usedinterchangeably unless otherwise clear from the context. Expressionvectors designed to produce RNAs as described herein in vitro or in vivomay contain sequences recognized by any RNA polymerase, includingmitochondrial RNA polymerase, RNA pol I, RNA pol II, and RNA pol III.These vectors can be used to transcribe the desired RNA molecule in thecell according to this invention. A plant transformation vector is to beunderstood as a vector suitable in the process of plant transformation.

Wild-type: The term “wild-type”, “natural” or “natural origin” meanswith respect to an organism, polypeptide, or nucleic acid sequence, thatsaid organism is naturally occurring or available in at least onenaturally occurring organism which is not changed, mutated, or otherwisemanipulated by man.

EXAMPLES

Chemicals and Common Methods

Unless indicated otherwise, cloning procedures carried out for thepurposes of the present invention including restriction digest, agarosegel electrophoresis, purification of nucleic acids, Ligation of nucleicacids, transformation, selection and cultivation of bacterial cells wereperformed as described (Sambrook et al., 1989). Sequence analyses ofrecombinant DNA were performed with a laser fluorescence DNA sequencer(Applied Biosystems, Foster City, Calif., USA) using the Sangertechnology (Sanger et al., 1977). Unless described otherwise, chemicalsand reagents were obtained from Sigma Aldrich (Sigma Aldrich, St. Louis,USA), from Promega (Madison, Wis., USA), Duchefa (Haarlem, TheNetherlands) or Invitrogen (Carlsbad, Calif., USA). Restrictionendonucleases were from New England Biolabs (Ipswich, Mass., USA) orRoche Diagnostics GmbH (Penzberg, Germany). Oligonucleotides weresynthesized by Eurofins MWG Operon (Ebersberg, Germany).

Example 1 Identification of Nucleic Acid Expression Enhancing NucleicAcids (NEENA) from Genes with Constitutive Expression

1.1 Identification of NEENA Molecules from A. thaliana Genes

Using publicly available genomic DNA sequences (e.g.ncbi.nlm.nih.gov/genomes/PLANTS/PlantList.html) and transcriptexpression data (e.g.wei-gelworld.org/resources/microarray/AtGenExpress/), a set of 18potential NEENA candidates deriving from Arabidopsis thalianatranscripts from highly expressing constitutive genes were selected fordetailed analyses. In addition, a putative NEENA molecule deriving fromparsley was also included in the analysis. The candidates were named asfollows:

TABLE 1 constitutive NEENA candidates (NEENAc). NEENA SEQ name LocusAnnotation ID NO NEENAc24 Petroselinum crispum gene Pcubi4-2 for 1polyubiquitin NEENAc17 At2g47170 ADP-ribosylation factor 1 (ARF1) 2NEENAc5 At1g56070 elongation factor 2, putative/EF-2, putative 3NEENAc18 At5g54760 eukaryotic translation initiation factor SUI1, 4putative NEENAc7 At4g02890 polyubiquitin (UBQ14) 5 NEENAc13 At3g03780AtMS2 (Arabidopsis thaliana methionine 6 synthase 2) NEENAc1 At5g60390elongation factor 1-alpha/EF-1-alpha 7 NEENAc21 At1g14400ubiquitin-conjugating enzyme 1 (UBC1) 8 NEENAc16 At4g14880 cysteinesynthase/O-acetylserine (thiol)-lyase/ 9 O-acetylserine sulfhydrylase(OAS1) NEENAc2 At4g27960 ubiquitin-conjugating enzyme E2-17 kDa 9 10(UBC9) NEENAc14 At1g64230 ubiquitin-conjugating enzyme, putative 11NEENAc4 At2g37270 40S ribosomal protein S5 (RPS5A) 12 NEENAc6 At4g05050polyubiquitin (UBQ11) 13 NEENAc8 At1g43170 60S ribosomal protein L3(RPL3A) 14 NEENAc11 At1g01100 60S acidic ribosomal protein P1 (RPP1A) 15NEENAc12 At5g04800 40S ribosomal protein S17 (RPS17D) 16 NEENAc19At4g34110 polyadenylate-binding protein 2 (PABP2) 17 NEENAc22 At2g34770fatty acid hydroxylase (FAH1) (anticipated IME 18 effect) NEENAc23At5g17920 cobalamin-independent methionine synthase 19 (CIMS)

1.2 Isolation of the NEENA Candidates

Genomic DNA was extracted from A. thaliana green tissue using the QiagenDNeasy Plant Mini Kit (Qiagen, Hilden, Germany). For the putative NEENAmolecule with the SEQ ID NO1, DNA of the vector construct 1bxPcUbi4-2GUS(WO 2003102198) was used. Genomic DNA fragments containing putativeNEENA molecules were isolated by conventional polymerase chain reaction(PCR). The polymerase chain reaction comprised 19 sets of primers (Table2). Primers were designed on the basis of the A. thaliana genomesequence with a multitude of NEENA candidates. The nucleotide sequenceof the vector construct 1bxPcUbi4-2GUS (WO 2003102198) was used for thedesign of primers (SEQ ID NO56 and 57) for amplification of the NEENAcandidate with SEQ ID NO1 (Table 2). The polymerase chain reactionfollowed the protocol outlined by Phusion High Fidelity DNA Polymerase(Cat No F-540L, New England Biolabs, Ipswich, Mass., USA). The isolatedDNA was used as template DNA in a PCR amplification using the followingprimers:

TABLE 2 Primer sequences SEQ PCR yield- ID ing SEQ ID Primer nameSequence NO NO NEENAc1_for tttatggtaccagccgcaagactcctttcagattct 20 7NEENAc1_rev aaattccatggtagctgtcaaaacaaaaacaaaaatcga 21 NEENAc2_foraaaaaggtacctcgaagaaccaaaaccaaaaacgtga 22 10 NEENAc2_revtttttccatggttatttatccaaaatcccacgatccaaattcca 23 NEENAc4_fortttttggtaccgatccctacttctctcgacact 24 12 NEENAc4_revttttaccatggtgactggaggatcaatagaagat 25 NEENAc5_fortttttggtacctttctctcgttctcatctttctctct 26 3 NEENAc5_revtaatagatatctttgtcaaacttttgattgtcacct 27 NEENAc6_fortataaggtaccaaatcaatctctcaaatctctca 28 13 NEENAc6_revtttatccatggtctgttaatcagaaaaaccgagat 29 NEENAc7_fortatatggtaccaaatcgttctttcaaatctctca 30 5 NEENAc7_revttataccatggtctgtaattcacaaaaaactgaga 31 NEENAc8_fortttttggtacctcatcgttggagcttagaagc 32 14 NEENAc8_revtttttccatggtcttcttcttcttcttctacatca 33 NEENAc11_fortatatggtaccaaagcattttcgatcttactcttaggt 34 15 NEENAc1l_revtttttccatggttttttatcctgaaacgattca 35 NEENAc12_fortttttggtaccttttgacgccgccgcttcttcttct 36 16 NEENAc12_revtttttccatggtctttcagttacctgtgtgacttacct 37 NEENAc13_fortttaaggtacccatctctcatctccactcttct 38 6 NEENAc13_revtttttgatatcttttgtttgttttttgtttttttact 39 NEENAc14_forttataggtaccaagtgaatcgtcaaaaccgagtt 40 11 NEENAc14_revtttttccatggttctcaaccaaaaaaaaactcct 41 NEENAc16_fortttttggtaccacgattcgggtcaaggttattga 42 9 NEENAc16_revtttttccatggtgattcaagcttcactgcttaaattcaca 43 NEENAc17_fortttttggtaccttagatctcgtgccgtcgtgcga 44 2 NEENAc17_revtttttccatggtttgatcaagcctgttcaca 45 NEENAc18_foraaaaaggtacctcatcagatcttcaaaaccccaa 46 4 NEENAc18_revaaaaaccatggtgatttgagggtagtactaaccgggaa 47 NEENAc19_forttttaggtaccatacgttaacttcaccaatccccaa 48 17 NEENAc19_revtttttccatggttaattaatgcagtgctttgtggtcgatgga 49 NEENAc21_fortttttcccgggatctttacctcaacaacgagat 50 8 NEENAc21_revtttttccatggttatcctcctttctttctaataaacaaaaccca 51 NEENAc22_fortttttggtacctctcttccgtctcgagtcgctgaga 52 18 NEENAc22_revtttttccatggtttgcagaccttttactgat 53 NEENAc23_fortttttggtaccttccttcctcctctccgattcttcct 54 19 NEENAc23_revtttttccatggttattgattttcttttactgcat 55 NEENAc24_forttttttggtaccttaagaaatcctctcttctcct 56 1 NEENAc24_revttttttccatggtctgcacatacataacatatca 57

Amplification during the PCR was carried out with the followingcomposition (50 microl):

3.00 microl A. thaliana genomic DNA (50 ng/microl genomic DNA, 5ng/microl vector construct)

10.00 microl 5×Phusion HF Buffer

4.00 microl dNTP (2.5 mM)

2.50 microl for Primer (10 microM)

2.50 microl rev Primer (10 microM)

0.50 microl Phusion HF DNA Polymerase (2 U/microl)

A touch-down approach was employed for the PCR with the followingparameters: 98.0° C. for 30 sec (1 cycle), 98.0° C. for 30 sec, 56.0° C.for 30 sec and 72.0° C. for 60 sec (4 cycles), 4 additional cycles eachfor 54,0° C., 51.0° C. and 49.0° C. annealing temperature, followed by20 cycles with 98.0° C. for 30 sec, 46.0° C. for 30 sec and 72.0° C. for60 sec (4 cycles) and 72.0° C. for 5 min. The amplification products wasloaded on a 2% (w/v) agarose gel and separated at 80V. The PCR productswere excised from the gel and purified with the Qiagen Gel ExtractionKit (Qiagen, Hilden, Germany). Following a DNA restriction digest withKpnI (10 U/microl) and NcoI (10 U/microl) or EcoRV (10U/microl)restriction endonuclease, the digested products were again purified withthe Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany).

1.3 Vector Construction

1.3.1 Generation of Vector Constructs with Potential NEENA Molecules

Using the Multisite Gateway System (Invitrogen, Carlsbad, Calif., USA),the promoter::NEENA::reporter-gene cassettes were assembled into binaryconstructs for plant transformation. The A. thaliana p-AtNit1(At3g44310, GenBank X86454; WO03008596, with the prefix p- denotingpromoter) promoter was used in the reporter gene construct, and fireflyluciferase (Promega, Madison, Wis., USA) was utilized as reporterprotein for quantitatively determining the expression enhancing effectsof the putative NEENA molecules to be analyzed.

The pENTR/A vector holding the p-AtNit1 promoter was cloned via sitespecific recombination (BP-reaction) between the pDONR/A vector andp-AtNit1 amplification products with primers p-AtNit1-for andp-AtNit1-rev (Table 3) on genomic DNA (see above) with site specificrecombination sites at either end according to the manufacturers manual(Invitrogen, Carlsbad, Calif., USA). Positive pENTR/A clones underwentsequence analysis to ensure correctness of the p-AtNit1 promoter.

TABLE 3 Primer sequences (p-AtNit1) SEQ ID Primer name Sequence NO.p-AtNit1-for ggggacaactttgtatagaaaagttgtcgaga 58 ccagatgttttacacttgap-AtNit1-rev ggggactgcttttttgtacaaacttggacact 59 cagagacttgagagaagca

An ENTR/B vector containing the firefly luciferase coding sequence(Promega, Madison, Wis., USA) followed by the t-nos nopalin synthasetranscriptional terminator (Genbank V00087) was generated. NEENAcandidate PCR fragments (see above) were cloned separately upstream ofthe firefly luciferase coding sequence using KpnI and NcoI or EcoRVrestriction enzymes. The resulting pENTR/B vectors are summarized intable 4, with promoter molecules having the prefix p-, coding sequenceshaving the prefix c-, and terminator molecules having the prefix t-.

TABLE 4 all pENTR/B vectors plus and minus NEENA candidates pENTR/BComposition of the partial expression cassette vector SEQ IDNO::reporter gene::terminator LJK1 MCS::c-LUC::t-nos LJK4 SEQ IDNO1::c-LUC::t-nos LJK40 SEQ ID NO7::c-LUC::t-nos LJK41 SEQ IDNO10::c-LUC::t-nos LJK43 SEQ ID NO12::c-LUC::t-nos LJK44 SEQ IDNO3::c-LUC::t-nos LJK46 SEQ ID NO13::c-LUC::t-nos LJK47 SEQ IDNO5::c-LUC::t-nos LJK48 SEQ ID NO14::c-LUC::t-nos LJK51 SEQ IDNO15::c-LUC::t-nos LJK52 SEQ ID NO16::c-LUC::t-nos LJK53 SEQ IDNO6::c-LUC::t-nos LJK54 SEQ ID NO11::c-LUC::t-nos LJK56 SEQ IDNO9::c-LUC::t-nos LJK57 SEQ ID NO2::c-LUC::t-nos LJK58 SEQ IDNO4::c-LUC::t-nos LJK59 SEQ ID NO17::c-LUC::t-nos LJK61 SEQ IDNO8::c-LUC::t-nos LJK62 SEQ ID NO18::c-LUC::t-nos LJK63 SEQ IDNO19::c-LUC::t-nos

The pENTR/C vector was constructed by introduction of a multiple cloningsite (SEQ ID N060) via KpnI and HindIII restriction sites. By performinga site specific recombination (LR-reaction), the created pENTR/A,pENTR/B and pENTR/C were combined with the pSUN destination vector (pSUNderivative) according to the manufacturers (Invitrogen, Carlsbad,Calif., USA) Multisite Gateway manual. The reactions yielded 1 binaryvector with p-AtNit1 promoter, the firefly luciferase coding sequencec-LUC and the t-nos terminator and 19 vectors harboring SEQ ID NO1, NO2,NO3, NO4, NO5, NO6, NO7, NO8, N09, NO10, NO11, NO12, NO13, NO14, NO15,NO16, NO17, NO18 and NO19 immediately upstream of the firefly luciferase coding sequence (Table 5), for which the combination with SEQ IDNO1 is given exemplary (SEQ ID NO61). Except for varying SEQ ID NO2 toNO19, the nucleotide sequence is identical in all vectors (Table 5). Theresulting plant transformation vectors are summarized in table 5:

TABLE 5 Plant expression vectors for A. thaliana transformationComposition of the expression cassette SEQ plant expressionPromoter::SEQ ID NO::reporter ID vector gene::terminator NO LJK132p-AtNit1::-::c-LUC::t-nos LJK133 p-AtNit1::SEQ ID NO1::c-LUC::t-nos 61LJK91 p-AtNit1::SEQ ID NO7::c-LUC::t-nos LJK92 p-AtNit1::SEQ IDNO10::c-LUC::t-nos LJK94 p-AtNit1::SEQ ID NO12::c-LUC::t-nos LJK95p-AtNit1::SEQ ID NO3::c-LUC::t-nos LJK97 p-AtNit1::SEQ IDNO13::c-LUC::t-nos LJK98 p-AtNit1::SEQ ID NO5::c-LUC::t-nos LJK99p-AtNit1::SEQ ID NO14::c-LUC::t-nos LJK102 p-AtNit1::SEQ IDNO15::c-LUC::t-nos LJK103 p-AtNit1::SEQ ID NO16::c-LUC::t-nos LJK104p-AtNit1::SEQ ID NO6::c-LUC::t-nos LJK105 p-AtNit1::SEQ IDNO11::c-LUC::t-nos LJK107 p-AtNit1::SEQ ID NO9::c-LUC::t-nos LJK108p-AtNit1::SEQ ID NO2::c-LUC::t-nos LJK109 p-AtNit1::SEQ IDNO4::c-LUC::t-nos LJK110 p-AtNit1::SEQ ID NO17::c-LUC::t-nos LJK112p-AtNit1::SEQ ID NO8::c-LUC::t-nos LJK113 p-AtNit1::SEQ IDNO18::c-LUC::t-nos LJK114 p-AtNit1::SEQ ID NO19::c-LUC::t-nos

The resulting vectors were subsequently used to transform A. thalianaleaf protoplasts transiently.

1.3.2 Renilla luciferase Control Construct

Renilla luciferase cDNA was amplified using 10 ng of the plasmidpRL-null from Promega (Madison, Wis., USA) as DNA template and primersR-LUC_for and R-LUC_rev (Table 6) with PCR parameters as describedabove.

TABLE 6 Primer sequences (c-RLUC) Primer SEQ ID name Sequence NORLUC_for aaaaaggtaccatgacttcgaaagtttatgatc 62 RLUC_revaaattgagctcttattgttcatttttgagaactc 63

Following a DNA restriction digest with KpnI (10 U/microl) and SacI (10U/microl) restriction endonuclease, the digested products were againpurified with the Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany).

The fragment was cloned into a ENTR/B vector containing the nopalinesynthase constitutive promoter p-nos (Genbank V00087) followed by thet-nos nopalin synthase transcriptional terminator (Genbank V00087) viaKpnI and SacI restriction sites, yielding a pENTR/B clone, whichunderwent sequence analysis to ensure correctness of the Renillaluciferase containing expression cassette.

Example 2 Screening for NEENA Candidate Molecules Enhancing GeneExpression in A. thaliana Transiently Transformed Leaf Protoplasts

This example illustrates that only selected NEENA molecules are capableof enhancing gene expression.

2.1 Isolation and Transient Transformation of A. thaliana LeafProtoplasts

Isolation and transient transformation of A. thaliana leaf protoplastswas amended according to established protocols (Damm and Willmitzer,1988; Damm et al., 1989) Leaves of 4 week old A. thaliana plants werecut in small pieces using a razor blade and transferred to a solutionwith 1.5% Cellulase R10 (Duchefa, Haarlem, The Netherlands), 0.3%Mazerozyme R10 (Duchefa, Haarlem, The Netherlands), 400 mM Mannitol, 20mM KCl, 20 mM MES, 10 mM CaCl₂, pH5.7 and incubated over night at roomtemperature. Due to a variability of transient A. thaliana leafprotoplast transformation, Renilla luciferase (Dual-Luciferase® ReporterAssay System, Promega, Madison, Wis., USA) was used to normalize thefirefly luciferase expression capabilities of the constructs above. Thetransient transformation of the NEENA-less (LJK132) and eachNEENA-containing vector construct (LJK66-LJK114) was performed intriplicate with 6 microg plasmid DNA, which was mixed with 25 microg ofRenilla luciferase containing construct prior to transformation, usingPEG (poly ethylene glycol) and 1×10⁴ protoplasts.

2.2 Dual luciferase Reporter Gene Assay

Transfected A. thaliana protoplasts were collected by centrifugation at100 g and frozen in liquid nitrogen after removal of supernatant. Theassay for detection of firefly and Renilla luciferase activity in thetransfected cells was performed according to the manufacturers (Promega,Madison, Wis., USA) Dual-Luciferase Reporter Assay System manual.Luminescence measurements were conducted in a MicroLumat Plus LB96V(Berthold Technologies, Bad Wildbad, Germany) recorded after addition ofthe luciferase substrates. Instrument readings of both luciferaserecordings were normalized by generating a ratio between fireflyluciferase and Renilla luciferase. The data from three experiments wereaveraged for each construct and based on these average expressionvalues, fold change values were calculated to assess the impact ofpresence of a putative NEENA over reporter gene constructs lacking therespective putative NEENA. In comparison to p-AtNit1 promoter-onlyNEENA-less reporter gene constructs, the 19 tested NEENA candidatescontaining constructs showed negative as well as positive effects,ranging from 0.1-fold to 18.1-fold induction in firefly Luciferaseactivity (FIG. 1). In total, 9 putative NEENA molecules comprisingsequences with SEQ ID NO1, NO2, NO3, NO4, NO5, NO6, NO7, NO8 and NO9conferred a greater than 2-fold increase in gene expression based onluciferase reporter gene activity compared to the NEENA-lesspromoter-only reporter gene construct (FIG. 1) and hence are functionalNEENA molecules. Since a number of the tested NEENA candidate moleculeshave marginal or even negative effects on the enhancement of geneexpression, not all putative NEENA molecules are mediating a commonstimulatory effect, but rather that the selected NEENA sequences conveysignificant enhancement of gene expression (SEQ ID NO. 1 to 9).

Example 3 Test of NEENA Molecules for Enhancement of Gene Expression inOilseed Rape Plants

This example illustrates that NEENA molecules can be used across speciesto enhance gene expression in all tissues tested compared to aNEENA-less promoter-only approach.

NEENA molecules mediating the strongest enhancement in gene expressionin the prescreening (cp. Example 2, SEQ ID NO1, NO2, NO3, NO4 and NO5)were selected for determining the enhancement on gene expression levelsin transgenic oilseed rape plants.

3.1 Vector Construction for B. napus Plant Transformation

For transformation of oilseed rape plants, reporter gene expressioncassettes without and with gene expression control molecules (SEQ IDsNO1-NO5) were combined with a gene expression cassette carrying aselectable marker gene for detecting transgenic plant lines within apENTR/C vector. By performing a site specific recombination(LR-reaction), as previously described (see above, 1.4), the pENTR/A,pENTR/B and the pENTR/C carrying the selectable marker cassette werecombined with the pSUN destination vector according to the manufacturers(Invitrogen, Carlsbad, Calif., USA) Multisite Gateway manual. Thereactions yielded one binary vector with p-AtNit1 promoter, the fireflyluciferase coding sequence c-LUC, the t-nos terminator and theselectable marker cassette as well as 5 vectors harboring SEQ ID NO1,NO2, NO3, NO4, and NO5 immediately upstream of the firefly luciferasecoding sequence (Table 7), for which the combination with SEQ ID NO1 isgiven exemplary (SEQ ID NO64). Except for varying SEQ ID NO2 to NO5, thenucleotide sequence is identical in all vectors (Table 7). The resultingplant transformation vectors are summarized in table 7:

TABLE 7 Plant expression vectors for B. napus transformation plant SEQexpression Composition of the expression cassette ID vectorPromoter::SEQ ID NO::reporter gene::terminator NO LJK138p-AtNit1::-::c-LUC::t-nos LJK139 p-AtNit1::SEQ ID NO1::c-LUC::t-nos 64LJK141 p-AtNit1::SEQ ID NO3::c-LUC::t-nos LJK142 p-AtNit1::SEQ IDNO5::c-LUC::t-nos LJK143 p-AtNit1::SEQ ID NO2::c-LUC::t-nos LJK144p-AtNit1::SEQ ID NO4::c-LUC::t-nos

3.2 Generation of Transgenic Rapeseed Plants (Amended Protocol Accordingto Moloney et al., 1992, Plant Cell Reports, 8: 238-242).

In preparation for the generation of transgenic rapeseed plants, thebinary vectors were transformed into Agrobacterium tumefaciensC58C1:pGV2260 (Deblaere et al., 1985, Nucl. Acids. Res. 13: 4777-4788).A 1:50 dilution of an overnight culture of Agrobacteria harboring therespective binary construct was grown in Murashige-Skoog Medium(Murashige and Skoog, 1962, Physiol. Plant 15, 473) supplemented with 3%saccharose (3MS-Medium). For the transformation of rapeseed plants,petioles or hypocotyledons of sterile plants were incubated with a 1:50Agrobacterium solution for 5-10 minutes followed by a three-daycoincubation in darkness at 25° C. on 3 MS. Medium supplemented with0.8% bacto-agar. After three days, the explants were transferred toMS-medium containing 500 mg/l Claforan (Cefotaxime-Sodium), 100 nMImazetapyr, 20 microM Benzylaminopurin (BAP) and 1.6 g/l Glucose in a 16h light/8 h darkness light regime, which was repeated in weekly periods.Growing shoots were transferred to MS-Medium containing 2% saccharose,250 mg/l Claforan and 0.8% Bacto-agar. After 3 weeks, the growth hormone2-Indolbutyl acid was added to the medium to promote root formation.Shoots were transferred to soil following root development, grown fortwo weeks in a growth chamber and grown to maturity in greenhouseconditions.

3.3 Plant Analysis

Tissue samples were collected from the generated transgenic plants fromleaves, flowers and siliques, stored in a freezer at −80° C. subjectedto a Luciferase reporter gene assay (amended protocol after Ow et al.,1986). After grinding the frozen tissue samples were resuspended in 800microl of buffer I (0.1 M Phosphate buffer pH7,8, 1 mM DTT (SigmaAldrich, St. Louis, Mo., USA), 0.05% Tween 20 (Sigma Aldrich, St. Louis,Mo., USA)) followed by centrifugation at 10 000 g for 10 min. 75 microlof the aqueous supernatant were transferred to 96-well plates. Afteraddition of 25 microl of buffer II (80 mM gycine-glycyl (Carl Roth,Karlsruhe, Germany), 40 mM MgSO4 (Duchefa, Haarlem, The Netherlands), 60mM ATP (Sigma Aldrich, St. Louis, Mo., USA), pH 7.8) and D-Luciferin toa final concentration of 0.5 mM (Cat No: L-8220, BioSynth, Staad,Switzerland), luminescence was recorded in a MicroLumat Plus LB96V(Berthold Technologies, Bad Wildbad, Germany) yielding the unit relativelight unit RLU per minute (RLU/min).

In order to normalize the luciferase activity between samples, theprotein concentration was determined in the aqueous supernatant inparallel to the luciferase activity (adapted from Bradford, 1976, Anal.Biochem. 72, 248). 5 microl of the aqueous cell extract in buffer I weremixed with 250 microl of Bradford reagent (Sigma Aldrich, St. Louis,Mo., USA), incubated for 10 min at room temperature. Absorption wasdetermined at 595 nm in a plate reader (Thermo Electron Corporation,Multiskan Ascent 354). The total protein amounts in the samples werecalculated with a previously generated standard concentration curve.Values resuiting from a ratio of RLU/min and mg protein/ml sample wereaveraged for transgenic plants harboring identical constructs and foldchange values were calculated to assess the impact of NEENA moleculepresence over NEENA-less reporter gene constructs.

3.4 NEENA Sequences Mediate Strong Enhancement of Gene Expression inOilseed Rape Plants

For assessing the potential of enhancing gene expression of selectedNEENA molecules (SEQ ID NO:1, 2, 3, 4 and 5) in oilseed rape plants,leafs, flowers and siliques harboring seeds of plants having identicaldevelopmental stages and which were grown under equal growth conditionswere collected. The samples were taken from individual transgenicoilseed rape plant lines harboring either a promoter-only reporter geneconstruct or Luciferase reporter gene constructs containing a NEENA (SEQID NO1, 2, 3, 4 and 5). 10 seeds were collected from each transgenicevent, processed and analyzed for Luciferase activity as described above(Example 3.3).

In comparison to the constitutive p-AtNit1 promoter-only NEENA-lessreporter gene construct, the five tested NEENA molecules all mediatedstrong enhancements in gene expression in leaf tissues (FIG. 2, a).Comparable enhancement of gene expression mediated by NEENAs (SEQ IDNO1, 2, 3, 4 and 5) was detected in oilseed rape flowers and siliquesincluding seeds (FIG. 2, b and c).

Example 4 Analysis of Constitutive Enhancement of Gene Expression inSoybean Plants

This example illustrates that NEENA molecules can be used in a widearray of plant species and across species borders from different plantfamilies to enhance gene expression in all tissues compared to aNEENA-less promoter-only approach.

NEENA sequence molecules mediating the strongest enhancement in geneexpression in the pre-screening (cp. Example 2, SEQ ID NO1, 2, 3, 4 and5) were selected for determining the enhancement on gene expressionlevels in transgenic soybean plants. Plant expression vectors LJK138,LJK139, LJK141, LJK142, LJK143 and LJK144 (cp. example 3.1) were usedfor stable soybean transformation.

4.1 Generation of Transgenic Soybean Plants (Amended Protocol Accordingto WO2005/121345; Olhoft et al., 2007).

Soybean seed germination, propagation, A. rhizogenes and axillarymeristem explant preparation, and inoculations were done as previouslydescribed (WO2005/121345; Olhoft et al., 2007) with the exception thatthe constructs LJK138, LJK139, LJK141, LJK142, LJK143 and LJK144 (cp.example 3.1) each contained a mutated AHAS gene driven by the parsleyubiquitin promoter PcUbi4-2, mediating tolerance to imidazolinoneherbicides for selection.

4.2 NEENA Sequences Mediate Strong Enhancement of Gene Expression inSoybean Plants

Tissue samples were collected from the generated transgenic plants fromleaves, flowers and seeds. The tissue samples were processed andanalyzed as described above (cp. example 3.3)

In comparison to the constitutive p-AtNit1 promoter-only NEENA-lessreporter gene construct, the five tested NEENA molecules all mediatedstrong enhancements in gene expression in leaves (FIG. 3a ). Comparableenhancement of gene expression mediated by NEENAs (SEQ ID NO1 to NO5)was detected in soybean flowers and siliques (FIG. 3, b and c).

Example 5 Analysis of NEENA Activity in Monocotyledonous Plants

This example describes the analysis of NEENA sequences with SEQ ID NO 1,2, 3, 4 and 5 in monocotyledonous plants.

5.1 Vector Construction

For analyzing NEENA sequences with SEQ ID NO 1, 2, 3, 4 and 5 inmonocotyledonous plants, a pUC-based expression vector harboring anexpression cassette composed of the NEENA-less, constitutivemonocotyledonous promoter p-Ubi from Z. mais is combined with a codingsequence of the beta-Glucuronidase (GUS) gene followed by the nopalinesynthase (NOS) transcriptional terminator. Genomic DNA is extracted fromA. thaliana green tissue using the Qiagen DNeasy Plant Mini Kit (Qiagen,Hilden, Germany). Genomic DNA fragments containing NEENA molecules areisolated by conventional polymerase chain reaction (PCR). Primers aredesigned on the basis of the A. thaliana genome sequence with amultitude of NEENA candidates. The reaction comprises 5 sets of primers(Table 8) and follows the protocol outlined by Phusion High Fidelity DNAPolymerase (Cat No F-540L, New England Biolabs, Ipswich, Mass., USA)using the following primers:

TABLE 8 Primer sequences SEQ PCR yield- ID ing SEQ ID Primer nameSequence NO NO NEENAc5_forII tttttggcgcgcctttctctcgttctcatctttctctct 653 NEENAc5_revII taataggcgcgcctttgtcaaacttttgattgtcacct 66 NEENAc7_forIItatatggcgcgccaaatcgttctttcaaatctctca 67 5 NEENAc7_revIIttataggcgcgcctctgtaattcacaaaaaactgaga 68 NEENAc17_forIItttttggcgcgccttagatctcgtgccgtcgtgcga 69 2 NEENAc17_revIItttttggcgcgcctttgatcaagcctgttcaca 70 NEENAc18_forIIaaaaaggcgcgcctcatcagatcttcaaaaccccaa 71 4 NEENAc18_revIIaaaaaggcgcgcctgatttgagggtagtactaaccgggaa 72 NEENAc24_forIIttttttggcgcgccttaagaaatcctctcttctcct 73 1 NEENAc24_revIIttttttggcgcgccctgcacatacataacatatca 74

Amplification during the PCR and purification of the amplificationproducts is carried out as detailed above (example 1.2). Following a DNArestriction digest with Ascl (10 U/microl) restriction endonuclease, thedigested products are purified with the Qiagen Gel Extraction Kit(Qiagen, Hilden, Germany).

NEENA PCR fragments (see above) are cloned separately upstream of thebeta-Glucuronidase coding sequence using Ascl restriction sites. Thereaction yields one binary vector with the p-Ubi promoter, thebeta-Glucuronidase coding sequence c-GUS and the t-nos terminator andfive vectors harboring SEQ ID NO1, NO2, NO3, NO4 and NO5, immediatelyupstream of the beta-Glucuronidase coding sequence (Table 9), for whichthe combination with SEQ ID NO1 is given exemplary (SEQ ID NO75). Exceptfor varying SEQ ID NO2 to NO5, the nucleotide sequence is identical inall vectors (Table 9). The resulting vectors are summarized in table 9,with promoter molecules having the prefix p-, coding sequences havingthe prefix c-, and terminator molecules having the prefix t-.

TABLE 9 Plant expression vectors plant SEQ expression Composition of theexpression cassette ID vector Promoter::SEQ ID NO::reportergene::terminator NO RTP2940 p-Ubi::-::c-GUS::t-nos LJK361 p-Ubi::SEQ IDNO1::c-GUS::t-nos 75 LJK362 p-Ubi::SEQ ID NO2::c-GUS::t-nos LJK363p-Ubi::SEQ ID NO3::c-GUS::t-nos LJK364 p-Ubi::SEQ ID NO4::c-GUS::t-nosLJK365 p-Ubi::SEQ ID NO5::c-GUS::t-nos

The resulting vectors are used to analyze NEENA molecules in experimentsoutlined below (Example 5.2).

5.2 Analysis of NEENA Molecules Enhancing Gene Expression inMonocotyledonous Plant Tissues

These experiments are performed by bombardment of monocotyledonous planttissues or culture cells (Example 6.2.1), by PEG-mediated (or similarmethodology) introduction of DNA to plant protoplasts (Example 6.2.2),or by Agrobacterium-mediated transformation (Example 6.3.3). The targettissue for these experiments can be plant tissues (e.g. leaf tissue),cultured plant cells (e.g. maize Black Mexican Sweetcorn (BMS), or plantembryos for Agrobacterium protocols.

5.2.1 Transient Assay using Microprojectile Bombardment

The plasmid constructs are isolated using Qiagen plasmid kit (cat#12143). DNA is precipitated onto 0.6 microM gold particles (Bio-Rad cat#165-2262) according to the protocol described by Sanford et al. (1993)(Optimizing the biolistic process for different biological applications.Methods in Enzymology, 217: 483-509) and accelerated onto target tissues(e.g. two week old maize leaves, BMS cultured cells, etc.) using aPDS-1000/He system device (Bio-Rad). All DNA precipitation andbombardment steps are performed under sterile conditions at roomtemperature. Black Mexican Sweet corn (BMS) suspension cultured cellsare propagated in BMS cell culture liquid medium [Murashige and Skoog(MS) salts (4.3 g/L), 3% (w/v) sucrose, myo-inositol (100 mg/L), 3 mg/L2,4-dichlorophenoxyacetic acid (2.4-D), casein hydrolysate (1 g/L),thiamine (10 mg/L) and L-proline (1.15 g/L), pH 5.8]. Every week 10 mLof a culture of stationary cells are transferred to 40 mL of freshmedium and cultured on a rotary shaker operated at 110 rpm at 27° C. ina 250 mL flask.

60 mg of gold particles in a siliconized Eppendorf tube are resuspendedin 100% ethanol followed by centrifugation for 30 seconds. The pellet isrinsed once in 100% ethanol and twice in sterile water withcentrifugation after each wash. The pellet is finally resuspended in 1mL sterile 50% glycerol. The gold suspension is then divided into 50microL aliquots and stored at 4° C. The following reagents are added toone aliquot: 5 microL of 1 microg/microL total DNA, 50 microL 2.5 MCaCl₂, 20 microL 0.1 M spermidine, free base. The DNA solution isvortexed for 1 minute and placed at −80° C. for 3 min followed bycentrifugation for 10 seconds. The supernatant is removed. The pellet iscarefully resuspended in 1 mL 100% ethanol by flicking the tube followedby centrifugation for 10 seconds. The supernatant is removed and thepellet is carefully resuspended in 50 microL of 100% ethanol and placedat −80° C. until used (30 min to 4 hr prior to bombardment). If goldaggregates are visible in the solution the tubes are sonicated for onesecond in a waterbath sonicator just prior to use.

For bombardment, two-week-old maize leaves are cut into piecesapproximately 1 cm in length and placed ad-axial side up on osmoticinduction medium M-N6-702 [N6 salts (3.96 g/L), 3% (w/v) sucrose, 1.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), casein hydrolysate (100mg/L), and L-proline (2.9 g/L), MS vitamin stock solution (1 mL/L), 0.2M mannitol, 0.2 M sorbitol, pH 5.8]. The pieces are incubated for 1-2hours.

In the case of BMS cultured cells, one-week-old suspension cells arepelleted at 1000 g in a Beckman/Coulter Avanti J25 centrifuge and thesupernatant is discarded. Cells are placed onto round ash-free No 42Whatman filters as a 1/16 inch thick layer using a spatula. The filterpapers holding the plant materials are placed on osmotic induction mediaat 27° C. in darkness for 1-2 hours prior to bombardment. Just beforebombardment the filters are removed from the medium and placed onto on astack of sterile filter paper to allow the calli surface to partiallydry.

Each plate is shot with 6 microL of gold-DNA solution twice, at 1.800psi for the leaf materials and at 1.100 psi for the BMS cultured cells.To keep the position of plant materials, a sterilized wire mesh screenis laid on top of the sample. Following bombardment, the filters holdingthe samples are transferred onto M-N6-702 medium lacking mannitol andsorbitol and incubated for 2 days in darkness at 27° C. prior totransient assays.

The transient transformation via microprojectile bombardment of othermonocotyledonous plants are carried out using, for example, a techniquedescribed in Wang et al., 1988 (Transient expression of foreign genes inrice, wheat and soybean cells following particle bombardment. PlantMolecular Biology, 11 (4), 433-439), Christou, 1997 (Ricetransformation: bombardment. Plant Mol Biol. 35 (1-2)).

Expression levels of the expressed genes in the constructs describedabove (example 5.1) are determined by GUS staining, quantification ofluminescence/fluorescence, RT-PCR and protein abundance (detection byspecific antibodies) using the protocols in the art. GUS staining isdone by incubating the plant materials in GUS solution [100 mM NaHPO4,10 mM EDTA, 0.05% Triton X100, 0.025% X-Gluc solution (5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid dissolved in DMSO), 10% methanol, pH7.0] at 37° C. for 16-24 hours. Plant tissues are vacuum-infiltrated 2times for 15 minutes to aid even staining. Analyses of luciferaseactivities are performed as described above (example 2 and 3.3).

In comparison to the constitutive p-Ubi promoter-only NEENA-lessreporter gene construct, the NEENA molecules all mediate strongenhancement in gene expression in these assays.

5.2.2 Transient Assay Using Protoplasts

Isolation of protoplasts is conducted by following the protocoldeveloped by Sheen (1990) (Metabolic Repression of Transcription inHigher Plants. The Plant Cell 2 (10), 1027-1038). Maize seedlings arekept in the dark at 25° C. for 10 days and illuminated for 20 hoursbefore protoplast preparation. The middle part of the leaves are cut to0.5 mm strips (about 6 cm in length) and incubated in an enzyme solutioncontaining 1% (w/v) cellulose RS, 0.1% (w/v) macerozyme R10 (both fromYakult Honsha, Nishinomiya, Japan), 0.6 M mannitol, 10 mM Mes (pH 5.7),1 mM CaCl₂, 1 mM MgCl₂, 10 mM beta-mercaptoethanol, and 0.1% BSA (w/v)for 3 hr at 23° C. followed by gentle shaking at 80 rpm for 10 min torelease protoplasts. Protoplasts are collected by centrifugation at 100×g for 2 min, washed once in cold 0.6 M mannitol solution, centrifuged,and resuspended in cold 0.6 M mannitol (2×10⁶/mL).

A total of 50 microg plasmid DNA in a total volume of 100 microL sterilewater is added into 0.5 mL of a suspension of maize protoplasts (1 x 10⁶cells/mL) and mixed gently. 0.5 mL PEG solution (40% PEG 4,000, 100 mMCaNO₃, 0.5 mannitol) is added and pre-warmed at 70° C. with gentleshaking followed by addition of 4.5 mL M M solution (0.6 M mannitol, 15mM MgCl₂, and 0.1% MES). This mixture is incubated for 15 minutes atroom temperature. The protoplasts are washed twice by pelleting at 600rpm for 5 min and resuspending in 1.0 mL of MMB solution [0.6 Mmannitol, 4 mM Mes (pH 5.7), and brome mosaic virus (BMV) salts(optional)] and incubated in the dark at 25° C. for 48 hr. After thefinal wash step, the protoplasts are collected in 3 mL MMB medium, andincubated in the dark at 25° C. for 48 hr.

The transient transformation of protoplasts of other monocotyledonousplants are carried out using, for example, a technique described inHodges et al., 1991 (Transformation and regeneration of riceprotoplasts. Biotechnology in agriculture No. 6, Rice Biotechnology.International Rice Research Institute, ISBN: 0-85198-712-5) or Lee etal., 1990 (Transient gene expression in wheat (Triticum aestivum)protoplasts. Biotechnology in agriculture and forestry 13—Wheat.Springer Verlag, ISBN-10: 3540518096).

Expression levels of the expressed genes in the constructs describedabove (Example 5.1) are determined by GUS staining, quantification ofluminescence/fluorescence, RT-PCR or protein abundance (detection byspecific antibodies) using the protocols in the art. GUS staining isdone by incubating the plant materials in GUS solution [100 mM NaHPO4,10 mM EDTA, 0.05% Triton X100, 0.025% X-Gluc solution(5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid dissolved in DMSO),10% methanol, pH 7.0] at 37° C. for 16-24 hours. Analyses of luciferaseactivities are performed as described above (Example 2 and 3.3).

In comparison to the constitutive p-Ubi promoter-only NEENA-lessreporter gene construct, the NEENA molecules mediate strong enhancementin gene expression in these assays.

5.2.3 Transformation and Regeneration of Monocotyledonous Crop Plants

The Agrobacterium-mediated plant transformation using standardtransformation and regeneration techniques may also be carried out forthe purposes of transforming crop plants (Gelvin and Schilperoort, 1995,Plant Molecular Biology Manual, 2nd Edition, Dordrecht: Kluwer AcademicPubl. ISBN 0-7923-2731-4; Glick and Thompson (1993) Methods in PlantMolecular Biology and Biotechnology, Boca Raton: CRC Press, ISBN0-8493-5164-2). The transformation of maize or other monocotyledonousplants can be carried out using, for example, a technique described inU.S. Pat. No. 5,591,616. The transformation of plants using particlebombardment, polyethylene glycol-mediated DNA uptake or via the siliconcarbonate fiber technique is described, for example, by Freeling &Walbot (1993) “The maize handbook” ISBN 3-540-97826-7, Springer VerlagNew York).

Expression levels of the expressed genes in the constructs describedabove (Example 5.1) are determined by GUS staining, quantification ofluminescence or fluorescence, RT-PCR, protein abundance (detection byspecific antibodies) using the protocols in the art. GUS staining isdone by incubating the plant materials in GUS solution [100 mM NaHPO4,10 mM EDTA, 0.05% Triton X100, 0.025% X-Gluc solution (5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid dissolved in DMSO), 10% methanol, pH7.0] at 37° C. for 16-24 hours. Plant tissues are vacuum-infiltrated 2times for 15 minutes to aid even staining. Analyses of luciferaseactivities are performed as described above (Examples 2 and 3.3).

In comparison to the constitutive p-Ubi promoter-only NEENA-lessreporter gene constructs, the NEENA molecules mediate strong enhancementin gene expression in plants.

Example 6 Quantitative Analysis of NEENA Activity in Corn Plants

This example describes the analysis of NEENA sequences with SEQ ID NO 1and 2 in corn plants.

6.1 Vector Construction

For analyzing NEENA sequences with SEQ ID NO 1 and 2 in monocotyledonousplants quantitatively, a pUC-based expression vector harboring anexpression cassette composed of the NEENA-less, constitutivemonocotyledonous promoter p-Ubi from Z. mais was combined with a codingsequence of the firefly luciferase (LUC) gene (Promega, Madison, Wis.,USA) followed by the nopaline synthase (NOS) transcriptional terminator.Genomic DNA was extracted from A. thaliana green tissue using the QiagenDNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Genomic DNA fragmentscontaining NEENA molecules were isolated by conventional polymerasechain reaction (PCR). Primers were designed on the basis of the A.thaliana genome sequence with a multitude of NEENA candidates. Thereaction comprised 2 sets of primers (Table 10) and followed theprotocol outlined by Phusion High Fidelity DNA Polymerase (Cat NoF-540L, New England Biolabs, Ipswich, Mass., USA) using the followingprimers:

TABLE 10 Primer sequences SEQ PCR yield- ID ing SEQ ID Primer nameSequence NO NO NEENAc17_forIII atatacgcgtttagatctcgtgccgtcg 76 2NEENAc17_revIII atatggcgcgcctttgatcaagcctgttcaca 77 NEENAc24_forIIIatatacgcgtttaagaaatcctctcttctcctc 78 1 NEENAc24_revIIIatatggcgcgccctgcacatacataacatatcaagatc 79

Amplification during the PCR and purification of the amplificationproducts was carried out as detailed above (example 1.2). Following aDNA restriction digest with MluI (10 U/microl) and AscI (10 U/microl)restriction endonucleases, the digested products were purified with theQiagen Gel Extraction Kit (Qiagen, Hilden, Germany).

NEENA PCR fragments (see above) were cloned separately upstream of thefirefly luciferase coding sequence using AscI restriction sites. Thereaction yielded one binary vector with the p-Ubi promoter, the fireflyluciferase coding sequence c-LUC and the t-nos terminator and twovectors harboring SEQ ID NO1 and NO2, immediately upstream of thefirefly luciferase coding sequence (Table 11), for which the combinationwith SEQ ID NO1 is given exemplary (SEQ ID NO80). Except for varying SEQID NO2, the nucleotide sequence is identical in the vectors (Table 11).The resulting vectors are summarized in table 11, with promotermolecules having the prefix p-, coding sequences having the prefix c-,and terminator molecules having the prefix t-.

TABLE 11 Plant expression vectors plant SEQ expression Composition ofthe expression cassette ID vector Promoter::SEQ ID NO::reportergene::terminator NO LJK309 p-Ubi::-::c-LUC::t-nos LJK327 p-Ubi::SEQ IDNO1::c-LUC::t-nos 80 LJK326 p-Ubi::SEQ ID NO2::c-LUC::t-nos

The resulting vectors were used to analyze NEENA molecules inexperiments outlined below (Example 6.2).

6.2 Generation of Transgenic Maize Plants

Maize germination, propagation, A. tumefaciens preparation andinoculations were done as previously described (WO2006136596,US20090249514) with the exception that the constructs LJK309, LJK326 andLJK327 (cp. example 6.1) each contained a mutated AHAS gene driven bythe corn ubiquitin promoter p-Ubi, mediating tolerance to imidazolinoneherbicides for selection.

6.3 NEENA Sequences Mediate Strong Enhancement of Gene Expression inCorn Plants

Tissue samples were collected from the generated transgenic plants fromleaves and kernels. The tissue samples were processed and analyzed asdescribed above (cp. example 3.3)

In comparison to the constitutive p-Ubi promoter-only NEENA-lessreporter gene construct, the two tested NEENA molecules mediated strongenhancements in gene expression in leaves (FIG. 4a ). Comparableenhancement of gene expression mediated by NEENAs (SEQ ID NO1 to NO2)was detected in maize kernels (FIG. 4b ).

Example 7 Quantitative Analysis of NEENA Activity in Rice Plants

This example describes the analysis of NEENA sequences with SEQ ID NO 1in rice plants.

7.1 Vector Construction

For analyzing NEENA sequences with SEQ ID NO 1 in rice plantsquantitatively, pENTR/B vectors LJK1 and LJK4 (compare example 1.3) werecombined with a destination vector harboring the constitutive PRO0239upstream of the recombination site using site specific recombination(LR-reaction) according to the manufacturers (Invitrogen, Carlsbad,Calif., USA) Gateway manual. The reactions yielded one binary vectorwith PRO0239 promoter, the firefly lucif erase coding sequence c-LUC andthe t-nos terminator as well as 1 vector harboring SEQ ID NO1immediately upstream of the firefly luciferase coding sequence (Table12). The resulting vectors are summarized in table 12, with promotermolecules having the prefix p-, coding sequences having the prefix c-,and terminator molecules having the prefix t-.

TABLE 12 Plant expression vectors plant SEQ expression Composition ofthe expression cassette ID vector Promoter::SEQ ID NO::reportergene::terminator NO CD30963 p-PRO0239::-::c-LUC::t-nos CD30964p-PRO0239::SEQ ID NO1::c-LUC::t-nos —

The resulting vectors were used to analyze NEENA molecules inexperiments outlined below (Example 7.2).

7.2 Generation of Transgenic Rice Plants

The Agrobacterium containing the respective expression vector was usedto transform Oryza sativa plants. Mature dry seeds of the rice japonicacultivar Nipponbare were dehusked. Sterilization was carried out byincubating for one minute in 70% ethanol, followed by 30 minutes in 0.2%HgCl₂, followed by a 6 times 15 minutes wash with sterile distilledwater. The sterile seeds were then germinated on a medium containing2,4-D (callus induction medium). After incubation in the dark for fourweeks, embryogenic, scutellum-derived calli were excised and propagatedon the same medium. After two weeks, the calli were multiplied orpropagated by subculture on the same medium for another 2 weeks.Embryogenic callus pieces were sub-cultured on fresh medium 3 daysbefore co-cultivation (to boost cell division activity).

Agrobacterium strain LBA4404 containing the respective expression vectorwas used for co-cultivation. Agrobacterium was inoculated on AB mediumwith the appropriate antibiotics and cultured for 3 days at 28° C. Thebacteria were then collected and suspended in liquid co-cultivationmedium to a density (OD₆₀₀) of about 1. The suspension was thentransferred to a Petri dish and the calli immersed in the suspension for15 minutes. The callus tissues were then blotted dry on a filter paperand transferred to solidified, co-cultivation medium and incubated for 3days in the dark at 25° C. Co-cultivated calli were grown on2,4-D-containing medium for 4 weeks in the dark at 28° C. in thepresence of a selection agent. During this period, rapidly growingresistant callus islands developed. After transfer of this material to aregeneration medium and incubation in the light, the embryogenicpotential was released and shoots developed in the next four to fiveweeks. Shoots were excised from the calli and incubated for 2 to 3 weekson an auxin-containing medium from which they were transferred to soil.Hardened shoots were grown under high humidity and short days in agreenhouse.

Approximately 35 independent T0 rice transformants were generated forone construct. The primary transformants were transferred from a tissueculture chamber to a greenhouse. After a quantitative PCR analysis toverify copy number of the T-DNA insert, only single copy transgenicplants that exhibit tolerance to the selection agent were kept forharvest of T1 seed. Seeds were then harvested three to five months aftertransplanting. The method yielded single locus transformants at a rateof over 50% (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al.1994).

7.3 NEENA Sequences Mediate Strong Enhancement of Gene Expression inRice Plants

Tissue samples were collected from the generated transgenic plants fromleaves and kernels. The tissue samples were processed and analyzed asdescribed above (cp. example 3.3)

In comparison to the constitutive p-PRO239 promoter-only NEENA-lessreporter gene construct, the tested NEENA molecule (SEQ ID NO 1)mediated strong enhancements in gene expression in leaves (FIG. 5a ).Strong enhancement of gene expression mediated by the NEENA (SEQ ID NO1)was detected in rice seeds (FIG. 5b ).

What is claimed is:
 1. A method for production of a high expressionconstitutive plant promoter, comprising functionally linking to apromoter one or more nucleic acid expression enhancing nucleic acid(NEENA) molecule heterologous to said promoter, wherein said NEENAcomprises a nucleic acid molecule selected from the group consisting of:i) a nucleic acid molecule comprising the nucleic acid sequence of SEQID NO: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and19; ii) a nucleic acid molecule having at least 80% sequence identity toany of the nucleic acid molecules of SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18 and 19; iii) a fragment of 100 ormore consecutive bases of the nucleic acid molecule of i) or ii) whichhas the expression enhancing activity of the corresponding nucleic acidmolecule of SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18 and 19; iv) a nucleic acid molecule which is the complementor reverse complement of any of the nucleic acid molecules of i) or ii);v) a nucleic acid molecule which is obtained by PCR using any of theoligonucleotide primers of SEQ ID NO: 20 to 57; and vi) a nucleic acidmolecule that hybridizes to a nucleic acid molecule comprising at least50 consecutive nucleotides of any of the transcription enhancingnucleotide sequences of SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18 and 19, or the complement thereof, wherein thenucleic acid molecule hybridizes under conditions comprisinghybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, and 1 mMEDTA at 50° C. and washing in 2×X SSC, 0.1% SDS at 50° C.
 2. A methodfor producing a plant or part thereof with enhanced constitutiveexpression of one or more nucleic acid molecule compared to a respectivecontrol plant or part thereof, comprising: a) introducing into a plantor part thereof one or more NEENA comprising the nucleic acid moleculeof claim 1 i) to vi); and b) functionally linking said one or more NEENAto a constitutive promoter and to a nucleic acid molecule under thecontrol of said constitutive promoter, wherein the NEENA is heterologousto said nucleic acid molecule.
 3. The method of claim 1, comprising: a)introducing the one or more NEENA into a plant cell, plant, or partthereof; b) integrating said one or more NEENA into the genome of saidplant cell, plant, or part thereof, whereby said one or more NEENA isfunctionally linked to an endogenous constitutively expressed nucleicacid heterologous to said one or more NEENA; and optionally c)regenerating a plant or part thereof comprising said one or more NEENAfrom said transformed plant cell.
 4. The method of claim 1 comprising:a) providing an expression construct comprising the one or more NEENAfunctionally linked to a constitutive promoter and to one or moreheterologous nucleic acid molecule, wherein said heterologous nucleicacid molecule is heterologous to said one or more NEENA and is under thecontrol of said constitutive promoter; b) integrating said expressionconstruct comprising said one or more NEENA into the genome of saidplant or part thereof; and optionally c) regenerating a plant or partthereof comprising said one or more expression construct from saidtransformed plant or part thereof.
 5. The method of claim 1, wherein theplant is a monocot or dicot plant.
 6. The method of claim 5, wherein theplant is a dicot plant.
 7. The method of claim 5, wherein the plant is amonocot plant.
 8. The method of claim 4, wherein said one or more NEENAis functionally linked to a constitutive promoter close to thetranscription start site of said heterologous nucleic acid molecule. 9.The method of claim 8, wherein said one or more NEENA is functionallylinked to a constitutive promoter that is 2500 bp or fewer away from thetranscription start site of said heterologous nucleic acid molecule. 10.The method of claim 4, wherein said one or more NEENA is functionallylinked to a constitutive promoter that is upstream of the translationalstart site of the heterologous nucleic acid molecule, and the expressionof said heterologous nucleic acid molecule is under the control of saidconstitutive promoter.
 11. The method of claim 4, wherein said one ormore NEENA is functionally linked to a constitutive promoter within the5′ UTR of the heteologous nucleic acid molecule, and the expression ofsaid heterologous nucleic acid molecule is under the control of saidconstitutive promoter.
 12. A recombinant expression construct comprisingone or more NEENA comprising a nucleic acid molecule selected from thegroup consisting of: i) the nucleic molecule of SEQ ID NO: 1, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19; ii) a nucleicacid molecule having at least 80% sequence identity to any of thenucleic acid molecules of SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18 and 19; iii) a fragment of 100 or moreconsecutive bases of the nucleic acid molecule of i) or ii) which hasthe expression enhancing activity of the corresponding nucleic acidmolecule of SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18 and 19; iv) a nucleic acid molecule which is the complementor reverse complement of any of the nucleic acid molecules of i) or ii);v) a nucleic acid molecule which is obtained by PCR using any of theoligonucleotide primers of SEQ ID NO: 20 to 57; and vi) a nucleic acidmolecule that hybridizes to a nucleic acid molecule comprising at least50 consecutive nucleotides of any of the transcription enhancingnucleotide sequences of SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18 and 19, or the complement thereof, wherein thenucleic acid molecule hybridizes under conditions comprisinghybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, and 1 mMEDTA at 50° C. and washing in 2×SSC, 0.1% SDS at 50° C.
 13. Therecombinant expression construct of claim 12, wherein the one-or moreNEENA is functionally linked to one or more constitutive promoter andone or more expressed nucleic acid molecule, wherein the expressednucleic acid molecule is heterologous to said one or more NEENA.
 14. Arecombinant expression vector comprising one or more recombinantexpression construct of claim 12
 15. A transgenic plant cell, plant, orpart thereof comprising one or more heterologous NEENA comprising anucleic acid molecule selected from the group consisting of: i) thenucleic molecule of SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18 and 19; ii) a nucleic acid molecule having at least80% sequence identity to any of the nucleic acid molecules of SEQ ID NO:1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19; iii)a fragment of 100 or more consecutive bases of the nucleic acid moleculeof i) or ii) which has the expression enhancing activity of thecorresponding nucleic acid molecule of SEQ ID NO: 1, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19; iv) a nucleic acidmolecule which is the complement or reverse complement of any of thenucleic acid molecules of i) or ii); v) a nucleic acid molecule which isobtained by PCR using any of the oligonucleotide primers of SEQ ID NO:20 to 57; and vi) a nucleic acid molecule that hybridizes to a nucleicacid molecule comprising at least 50 consecutive nucleotides of any ofthe transcription enhancing nucleotide sequences of SEQ ID NO: 1, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19, or thecomplement thereof, wherein the nucleic acid molecule hybridizes underconditions comprising hybridization in 7% sodium dodecyl sulfate (SDS),0.5 M NaPO₄, and 1 mM EDTA at 50° C. and washing in 2×SSC, 0.1% SDS at50° C.
 16. A transgenic cell, plant, or part thereof comprising: a) therecombinant expression construct of claim 12; or b) a recombinantexpression vector comprising one or more of said recombinant expressionconstruct.
 17. The transgenic cell, of claim 16, selected or derivedfrom the group consisting of bacteria, fungi, yeasts, or plants.
 18. Thetransgenic plant or part thereof of claim 16, wherein said plant or partthereof is dicotyledonous.
 19. The transgenic plant or part thereof ofclaim 16, wherein said plant or part thereof is monocotyledonous.
 20. Atransgenic cell, cell culture, seed, plant, plant part, or propagationmaterial derived from the transgenic cell, plant, or part thereof ofclaim 16, wherein the transgenic cell, cell culture, seed, plant, plantpart, or propagation material comprises said NEENA.
 21. A method for theproduction of foodstuffs, animal feed, seeds, a pharmaceutical, or afine chemical comprising: a) providing a transgenic cell culture, seed,plant, plant part, or propagation material derived from the transgeniccell or plant of claim 20; and b) preparing foodstuffs, animal feed,seeds, a pharmaceutical, or a fine chemical from the transgenic cellculture, seed, plant, plant part, or propagation material of a).